ES2623287T3 - Highly active nucleoside derivative for HCV treatment - Google Patents

Abstract

A compound of Formula I or a pharmaceutically acceptable salt thereof, where Formula I is ** Formula ** where R1 and R2 are each hydrogen or D and each position represented as D has deuterium enrichment of at least 50%.

Description

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DESCRIPTION

Highly active nucleoside derivative for the treatment of HCV Background

It is estimated that 3% of the world's population is infected with the hepatitis C virus. The World Health Organization estimates that 150 million people are chronically infected worldwide. Of those exposed to HCV, 80% to 85% become chronically infected, at least 30% develop liver cirrhosis and 1-4% develop hepatocellular carcinoma. Hepatitis C Virus (HCV) is one of the most prevalent causes of chronic liver disease in the United States, as it represents approximately 15 percent of acute hepatitis vmca, 60 to 70 percent of hepatitis chronic and up to 50 percent of cirrhosis, liver disease in the terminal phase, and liver cancer. Chronic HCV infection is the most common cause of liver transplantation in the US, Australia and most of Europe. Hepatitis C is estimated to cause 10,000 to 12,000 deaths annually in the United States. Although the acute phase of HCV infection is usually associated with mild symptoms, some evidence suggests that only about 15% to 20% of infected people spontaneously eliminate HCV.

HCV is a single-enveloped helix RNA virus. HCV is classified as a member of the genus Hepacivirus of the Flaviviridae family. At least 4 strains of HCV, GT-1-GT-4 have been characterized. The HCV life cycle includes entry into host cells, the HCV genome translation, the processing of polyproteins and the assembly of the replicase complex, the replication of the RNA and the assembly and release of virions. In the RNA replication process, a complementary negative helix copy of the genomic RNA is produced. The negative helix copy is used as a template to synthesize additional positive helix genomic RNAs that can participate in the translation, replication, packaging or any combination of these to produce a virus progeny.

There are several proteins in hepatitis C that have been addressed for drug therapy. NS5A is a proline-rich hydrophilic phosphoprotem that binds zinc without inherent enzymatic activity, which can be inhibited with certain non-nucleotide compounds. NS5B is a key enzyme that plays the most important role in the replication of HCV viral RNA using a vmca positive RNA helix as a template, which has been inhibited with synthetic nucleoside derivatives. The NS2-3 protease is an enzyme responsible for proteolytic cleavage between NS2 and NS3, which are non-structural proteins. The NS3 protease is responsible for the cleavage of the nonstructural protein sequence below. RNA helicase uses the hydrolysis of ATP to unwind the RNA.

Sofosbuvir (Sovaldi, see the structure below) is an NS5B inhibitor nucleoside phosphoramidate approved in December 2013 for the treatment of HCV. The approved indications recommend the following regimes: (i) for genotypes 2 and 3, an oral tablet of 400 mg once daily in combination with ribavirin and (ii) for genotypes 1 and 4, an oral tablet of 400 mg a once a day (triple combination therapy) with ribavirin and pegylated interferon. Treatment with sofosbuvir lasts 12 weeks for genotypes 1, 2 and 4 and 24 weeks for genotype 3. Sofosbuvir can also be used with ribavirin for the treatment of patients who have chronic hepatitis C with hepatocellular carcinoma waiting for a liver transplant. for up to 48 weeks or until liver transplantation to prevent post-transplant HCV infection. The FDA granted Sovaldi Priority Review and the Advance Therapy designation based on data from several large-scale weather tests that indicated a vmca maintained response (RVM) of twelve weeks in 50-90 percent of participants In the tests. Patients who reach an "RVM12" are often considered cured.

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Alios BioPharma, Inc. licensed Vertex Pharmaceuticals Inc. for ALS-2200 for the development of hepatitis C treatment in June 2011. ALS-2200 is a mixture of diastereomers in a chiral phosphorus stereocenter. Vertex is developing a unique diastereomer, VX-135, and it is currently in Phase II heat tests. Although the companies have not disclosed the chemical structure of VX-135, they have said that it is an analog prodrug of the uridine nucleotide, and an inhibitor of NS5B. In 2013, the FDA placed VX-135 in a partial thermal suspension after three patients who had received high dosages of VX-135 had liver toxicity. Reducing the dose of a nucleotide inhibitor to avoid toxicity can sometimes also compromise or decrease efficacy. Vertex announced in January 2014 that VX-135 in combination with daclastavir (NS5A inhibitor from Bristol-Myers Squibb) had completed a Phase 2a test. In a treatment attempt analysis, the vmca response rate maintained four weeks after treatment completion (RVM4) was 83% (10 of 12) in individuals infected with genotype 1 not previously treated with the treatment they received 200 mg of VX-135 in combination with daclatasvir. One patient exhibited a serious adverse episode of vomiting / nausea. The rest of the eleven patients completed 12 weeks of treatment, for a completion rate of treatment (RVM4) of 91%.

Idenix Pharmaceuticals Inc. is developing IDX21437 for the treatment of hepatitis C, which is a nucleotide prodrug of uridine inhibitor of NS5B. The details of the chemical structure have not been published to date. In April 2014, Idenix announced that 300 mg of IDX21437 once a day for a period of six days resulted in a maximum average reduction in the vmca load of 4.2-4.3 log10 IU / ml in 18 patients who had not received the 1.2 or 3 genotype treatment.

Despite the progress in the area of hepatitis C treatment, there have also been a number of difficult setbacks. BMS-986094, a guanosine-based phosphoramidate for hepatitis C, was excluded from the clinical tests after the death of a patient due to heart failure in August 2012. BMS announced a continuation in 2013 that it was leaving the research area of the Hepatitis C. After the withdrawal of the BMS drug, the chemical suspension of the similar NS5B inhibitor of Idenix Pharmaceuticals, IDX 19368, which shares the same active metabolite, BMS-986094, was finally discontinued. This followed the previous chemical suspension and discontinuation of the development of the IDX184 nucleotide prodrug for the same indication.

It is known that effective treatment against hepatitis C includes combination therapy, due to the appearance of vmca resistance during monotherapy. Given the documented challenges of developing optimal agents for hepatitis C and the fact that multiple optimal agents are required for effective therapy, there is a strong need for additional agents for hepatitis C.

Several more antivmcos nucleoside derivatives are disclosed in WO2010 / 135569 A1 and WO2012 / 142523 A2.

Summary

Surprisingly, it has been found that the formula I, 2'-methyl-5'-deuterated uridine phosphoramidate, including Formula II, Formula IIA, Formula IIB, Formula IIIA or Formula IIIB, or a pharmaceutically acceptable salt thereof , where deuterium has an enrichment above hydrogen of at least 90%, and where R1 and R2 are independently deuterium or hydrogen, it is a superior inhibitor of NS5B for the treatment of hepatitis C. In one embodiment, R1 is deuterium and R2 is hydrogen.

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5 Therefore, in one embodiment, a method is provided for the treatment of a host infected with hepatitis C or another described aqm disorder that includes the administration of an effective amount of a compound of Formula I or II, or a pharmaceutically salt acceptable thereof, possibly on a pharmaceutically acceptable carrier.

In another embodiment, the nucleoside derivative of Formula I, II, IIIA, or IIIB is administered as an R or S stereoisomer of phosphorus, which is in at least 90% pure form, and typically in a 95, 98 or 99 form. % pure

One embodiment also includes a nucleoside derivative of Formula I, II, IIIA or IIIB, which is administered as a mixture of R or S stereoisomers of phosphorus, for example a 50/50 mixture. For example, a mixture, such as a 50/50 mixture, of Formula IIA and IIB can be administered.

In another embodiment, an effective amount of a compound of Formula IIIA or IIIB or its pharmaceutically acceptable salt, optionally on a pharmaceutically acceptable carrier, is provided to a host in need of hepatitis C therapy.

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When administered to the host, for example, Formula II phosphoramidate is metabolized to 5’-OH, 5’-D, D-monophosphate (Formula V) through a series of enzymatic stages.

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Hint 1

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Dephosphorylation

"* ■

Phosphorylation

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Formula VI

CatA: Cathepsin A CES1: Carboxiesterase 1

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Formula II, for example, becomes its active species, nucleoside triphosphate (Formula IV), through nucleoside monophosphate (Formula V). Alternatively, the nucleoside monophosphate (Formula V) may undergo dephosphorylation to 2'-C-methyluridine 5'-deuterated (Formula VI). 5'-deuterated nucleoside triphosphate (Formula IV) is the pharmacologically active metabolite that inhibits the replication of the hepatitis C virus, while 2'-C-methyluridine 5'-deuterated (Formula VI) shows little activity, since which is a poor substrate for the nucleoside monophosphate kinase.

10 5'-deuterated nucleoside monophosphate (Formula V), if dephosphorylated, will produce 5'-deuterated nucleoside (Formula VI) and non-deuterated nucleoside monophosphate (Formula VIII) will produce non-deuterated 2'-C-methyluridine (Formula IX).

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Formula VIII

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Formula IX

Surprisingly, it has been found that deuterium at the 5 'position of the nucleoside stabilizes the nucleoside derivative against dephosphorylation to the unwanted 5'-OH, 5'-deuterated nucleoside. This is surprising because the deuterium atom (s) do not cleave during dephosphorylation and do not bind to an atom that is cleaved during dephosphorylation. The disclosure includes the use of 5'-deuterium to produce a significant effect on metabolism and efficacy through a remote and unexpectedly important effect of the secondary deuterium isotope. Said important effect of the secondary deuterium isotope on the dephosphorylation in the 5 'position has not been previously reported. By increasing the stability of the nucleoside 5'-monophosphate against dephosphorylation, an increase in the nucleoside active 5'-triphosphate pool can be achieved, which may result in greater efficacy at a given oral dosage or equal efficiency using a lower dose of

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nucleoside in the climate. It can also have a significant effect on the half-life, and, therefore, on the pharmacokinetics, of the drug.

Therefore, in another embodiment, the present disclosure includes a method of treating an affected host with a disorder that can be treated with a nucleoside or nucleotide, the improvement consisting in replacing one or both of the hydrogen in the 5 'position of the nucleoside or nucleotide with a deuterium with at least 90% enrichment above the protium (i.e. less than 10% hydrogen 1H) (and in other embodiments an enrichment of 50, 95, 98 or 99%). The therapeutic effect of any nucleotide or nucleoside can be increased if the active metabolite is the mono-, di- or triphosphate of the nucleoside by 5 'deuteration of the nucleoside, since 5' deuteration increases the nucleoside monophosphate pool. Nucleoside monophosphate is metabolized to diphosphate and / or triphosphate with the corresponding nucleoside diphosphate kinase and then nucleoside triphosphate kinase. This method is especially useful for nucleosides that do not readily monophosphorylate and, therefore, lose substantial activity when dephosphorylated, which cannot be easily recovered by the action of nucleoside monophosphate kinase.

In one embodiment, the disorder is a vmca disease. In another embodiment, the disorder is hepatitis Bo C. In yet another embodiment, the disorder is HIV. In another embodiment, the disorder is an abnormal cell proliferation. In yet another embodiment, the disorder is a tumor or cancer. The nucleosphid derivative used in this improved method may be, for example, a phosphoramidate or other stabilized nucleotide prodrug that is metabolized to 5'-monophosphate, or it may be 5'-monophosphate itself. In one embodiment, the nucleospheric derivative contains a 2'-methyl, 2'-p-hydroxynucleoside. In yet another embodiment, the nucleospheric derivative contains a 2'-a-methyl, 2'-p-fluoronucleoside. In eventual embodiments, the methyl group may have one or more halogens, for example, fluorine (s).

Nucleoside triphosphate (NTP) is the active species that inhibits vmca replication in hepatocytes and its intrinsic levels and potency drive the efficacy of treatment.

A demonstration of the increase in the levels of nucleoside triphosphate caused by the use of 5 'deuteration in Example 13 is given below. In this Example, the compound of Formula II, as well as the corresponding non-deuterated compound (Formula VII), was incubated in fresh hepatocytes for 24 hours. The data shows that there is more dephosphorylated nucleoside (i.e. unwanted 5'-OH nucleoside) in the samples incubated with the non-deuterated phosphoramidate than with the 5'-deuterated phosphoramidate. Specifically, using 20 | iM of Formula II or its non-deuterated counterpart, Formula VII, (12-well plate (1 ml) with hepatocytes seeded at a rate of 0.67 million cells per well for 24 hours) a concentration is obtained 1.9 times (medium, that is, extracellular concentration) and 2.9 times (cellular extract, that is, intracellular) greater than 2'-non-deuterated dephosphorylated methyluridine (Formula IX) compared to the resulting 5 'form -deuterated (Formula VI). The results of the incubation of 20 | iM of Formula II or its non-deuterated counterpart (Formula VII) (6-well plate (2 ml) with hepatocytes seeded at the rate of 1.7 million cells per well for 24 hours) indicate a concentration 1.5 times (cellular extract, that is, intracellular) and 2.8 times (cellular extract, that is intracellular) greater than 2'-deuterated dephosphorylated methyluridine (Formula IX) compared to the resulting 5 'form -deuterated (Formula VI). Therefore, on average, dephosphorylation in hepatocytes leads to approximately double the 5'-OH-nucleoside produced when the 5 'position is not deuted. This difference in the 5'-monophosphate pool (such as Formula VIII for the non-deuterated version) available for triphosphate activation when using 5'-deuterated phosphoramidate can have a significant effect on efficacy, dosage, toxicity. and / or the pharmacokinetics of the drug.

As a comparison with a candidate for a chemical test, as will also be described in Example 14 and Figure 3, a poster presented by Alios (EASL 2013) indicates that the level of VX-135 triphosphate measured in human hepatocytes after 24 hours of incubation with 50 | iM of VX-135 it was 1,174 pmol / million cells. On the contrary, the level of Formula IV after 25 hours of incubation of human hepatocytes with 5 µM of Formula II, that is, a concentration ten times lower, is 486 pmol / million cells. Therefore, the amount of triphosphate produced by incubation of Formula II is 4 times higher (standardized dose) than the amount of triphosphate produced by VX-135. Although the precise structure of VX-135 is currently unknown, it is about an urotine nucleotide analog prodrug of NS5B inhibitor.

In addition, after incubation of Formula II at 50 nM in primary hepatocytes for 24 h, the level of Formula IV varied from 9.2 to 16.2 pmol / million cells. These concentrations are 5 to 8 times higher than those obtained when Huh-luc / neo cells were incubated at 50 nM. Since Formula IV is the active species that inhibits the replication of HCV replicons in Huh-luc / neo cells, the predicted EC50 of Formula II in primary human hepatocytes is 6.25-10 nM if HCV replicon could grow in primary hepatocytes, assuming that the linear relationship obtained in Figure 4 between Formula II and Formula IV continues at a lower concentration.

As detailed in Example 14 and Figure 5, the half-life of Formula IV is greater than the half-life of

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Sovaldi triphosphate in hepatocytes of four species. The greatest half-life was in human hepatocytes, followed by those of the dog, after those of the monkey and then of the rat. The half-lives range from 10 to 30 hours for Formula IV and from 8 to 23 hours for Sovaldi triphosphate.

In addition, as described in Example 17 and Figures 6 and 7, over a period of 48 hours, while the intracellular conversion to the corresponding Sovaldi triphosphate (GS-7977) measured in human hepatocytes is 2 times greater than that of the triphosphate derived from Formula II (i.e., Formula IV), the concentration of Formula IV continues to increase at 48 hours, while the concentration of the metabolite triphosphate of Sovaldi decreases from 24 to 48 hours. The increasing concentration of Formula IV, combined with its half-life of> 24 h, suggests accumulation of the levels of Formula IV (the formula II triphosphate) in hepatocytes after repeated dosing. This trend, after an initial dosing ramp in vivo until acclimatization is reached, can lead to a higher steady-state concentration of Formula IV in vivo for the triphosphate derived from Formula II than for Sovaldi triphosphate. In addition, the intrinsic potency (the inhibitory effect on the RdRp activity of NS5B) of Formula IV (the formula II triphosphate) is 1.5 times better than the intrinsic potency of Solvadi triphosphate.

The disclosure also includes a method of treating an HCV infection or a related disorder in a patient, consisting of providing a therapeutically effective amount of one or more of the active compounds described herein, optionally in combination or in alternation with one or more of other anti-HCV active ingredients or other medical therapies that offer an additive or synergistic benefit to the patient.

Brief description of the drawings

Fig. 1 is a Table showing the concentration of Formula IX (non-deuterated 2'-methyluridine 5'-OH) and Formula VI (5'-deuterated-5'-OH 2'-methyluridine) in hepatocyte medium human and in the cell extract after incubation with 20 µM of Formula VII or Formula II, respectively. Specifically, as described in Example 13, using 20 µM of Formula II or its non-deuterated Formula VII counterpart (12-well plate (1 ml) with hepatocytes seeded at a rate of 0.67 million cells per well for 24 hours) a concentration 1.9 times (medium, i.e. extracellular concentration) and 2.9 times (cell extract, i.e. intracellular) greater than 2'-deuterated dephosphorylated methyluridine (Formula IX) is obtained compared to the resulting from the 5'-deuterated form (Formula VI).

Fig. 2 is a Table showing the concentration of Formula IX (non-deuterated 2'-methyluridine 5'-OH) and Formula VI (5'-deuterated-5'-OH 2'-methyluridine) in hepatocyte medium human and in the cell extract after incubation with 20 µM of Formula VII or Formula II, respectively. As described in Example 13, the results of the incubation of 20 µM of Formula II or its counterpart of non-deuterated Formula VII (6-well plate (2 ml) with hepatocytes seeded at the rate of 1.7 million cells per well for 24 hours) indicate a concentration 1.5 times (cellular extract, that is, intracellular) and 2.8 times (cellular extract, that is, intracellular) greater than non-deuterated dephosphorylated 2'-methyluridine (Formula IX) in comparison with the result of the 5'-deuterated form (Formula VI).

Fig. 3 is a Table showing the concentrations of Formula IV (the active deuterated triphosphate metabolite of Formula II) produced in human hepatocytes (pmol of Formula IV / million cells) at 2, 4, 8, 25 or 48 hours of incubation with 5 | iM of Formula II. The results of three experiments are shown, together with the mean and standard deviation determined for each time point. Formula IV level peaks were obtained at> 48 hours in human hepatocytes. The concentration of VX-135-TP (the active triphosphate metabolite) generated from VX-135 at 24 hours is shown. As discussed in Example 14, the levels of triphosphate (Formula IV) generated from Formula II are 4 times higher than the levels of triphosphate (VX-135-TP) generated from VX-135, which suggests that VX-135 will be less powerful than Formula II.

Fig. 4 is a graph of the concentration of Formula IV (ng / ml) versus the concentration of Formula II (| iM) in human hepatocytes. Formula IV concentrations in human hepatocytes were determined after 24-hour incubations with 0.15, 0.45 and 1.35 µM of Formula II.

Fig. 5 is a Table showing the half-lives of active triphosphate (Formula IV or GS-7977-TP) in human, dog, monkey and rat hepatocytes. Formula II or GS-7977 (Sovaldi) was added at selected concentrations to hepatocytes (human, dog, monkey and rat) and incubated at 37 ° C. Cell extracts of the supernatant of Formula IV or GS-7977-TP (active triphosphate metabolites) were measured by high performance liquid chromatography with tandem mass spectrometric detection (LC-MS / MS). As discussed in Example 15, the values of the half-life of triphosphate varied between 8 and 30 hours and Formula IV generally had a half-life greater than GS-7977-TP across all the species studied. (a - Values in parentheses = 95% confidence interval).

Fig. 6 is a graph of the concentration of Formula IV (the active deuterated triphosphate metabolite of Formula II) produced in human hepatocytes (pmol of Formula IV / million cells) during 48 hours of incubation with 5 | iM of Formula II . Concentrations were measured at the indicated times and ABC was calculated with the Graphpad Prism 5 program. As discussed in Example 16, the peaks of Formula IV levels at> 48 hours were obtained in human hepatocytes.

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Fig. 7 is a graph of the concentration of GS-7977-TP (the Sovaldi active triphosphate metabolite) produced in human hepatocytes (pmol of GS-7977-TP / miNon of cells) during 48 hours of incubation with 5 | iM of GS-7977 (Sovaldi). The concentrations were measured at the indicated times and the ABC was calculated with the Graphpad Prism 5 program. As discussed in Example 16, the peaks of GS-7977-TP levels (the Sovaldi active triphosphate metabolite) were obtained. at 24 hours in human hepatocytes.

Detailed description

Chemical description and terminology

Compounds are described using standard nomenclature. Unless defined otherwise, all technical and scientific terms used by employees have the same meaning as that commonly understood by an expert in the technique to which this invention belongs.

The terms "a" and "a" do not denote a quantity limitation, but rather denote the presence of at least one of the referenced articles. The term "or" means "and / or". The non-limited connection expression "comprising" encompasses the intermediate connection expression "consisting essentially of" and the limited expression "consisting of". The citation of ranges of values is intended only to serve as an abbreviated method to refer individually to each independent value that remains within the range, unless something else is indicated here, and each independent value is incorporated into the specification as if it were cited here individually. . The valuation criteria of all ranks in the range are included and can be combined independently. All the methods described herein may be carried out in an appropriate order, unless otherwise indicated or that the context clearly contradicts it otherwise. The use of each and every one of the examples, or exemplary language (eg, "as is"), is only intended to illustrate and does not impose a limitation on the scope of the invention, unless otherwise claimed. No language should be considered in the specification as an indicator that any element not claimed is essential for the practice of the invention.

The compounds of Formula I include other formulas disclosed herein within the scope of Formula I. These include, for example, the compounds of Formula II, IIA, IIB and compound 22.

The compounds of Formula I include compounds of the formula that have isotopic substitutions in any position. Isotopes include those atoms that have the same atomic number, but different mass numbers. As a general example, and without limitation, hydrogen isotopes include tritium and deuterium and carbon isotopes include 11C, 13C and 14C. The compounds of Formula I also require enrichment of deuteration (substitution of a hydrogen atom with deuterium) at identified positions.

An "active ingredient" is a compound (including a compound disclosed herein), an element or mixture that, when administered to a patient, alone or in combination with another compound, element or mixture, confers, directly or indirectly, a physiological effect on the patient. The indirect physiological effect can occur through a metabolite or other indirect mechanism.

The term "substituted", as used herein, means that any one or more of the hydrogens on the atom or designated group is substituted with a selection from the indicated group, with the proviso that the normal valence of the atom is not exceeded. designated.

"Deuteration" and "deuterated" mean that a hydrogen in the specified position is replaced by deuterium. In any sample of a Formula I compound in which a position is deuterated, some discrete molecules of the Formula I compound are likely to have hydrogen, rather than deuterium, at the specified position. However, the percentage of molecules of the compound of Formula I in the sample that have deuterium in the specified position will be much higher than what appears naturally. Deuterium in the deuterated position is enriched. The term "enriched", as used herein, refers to the percentage of deuterium versus other species of hydrogen at that location. As an example, if a position in the compound of Formula I is said to contain 50% enrichment in deuterium, this means that more than hydrogen in the specified position the deuterium content is 50%. For reasons of clarity, it is confirmed that the term "enriched", as used here, does not mean enriched percentage above natural abundance. In one embodiment, the deuterated compounds of Formula I will have at least 10% enrichment in deuterium in any deuterated position. In other embodiments, there will be at least 50%, at least 90% or at least 95% enrichment in deuterium at the specified deuterated position or positions. A "deuterated substituent" is a substituent in which at least one hydrogen is replaced by deuterium at the specified enrichment percentage. "Eventually deuterated" means that the position can either be hydrogen and the amount of deuterium in the position is only the natural level of deuterium, or the position is enriched with deuterium above the natural level of deuterium.

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A "dosage form" means a unit of administration of an active ingredient. Non-limiting examples of dosage forms include tablets, capsules, injections, suspensions, fluids, intravenous fluids, emulsions, creams, ointments, suppositories, inhalable forms, transdermal forms and the like.

"Aryl" indicates aromatic groups containing only carbon in the aromatic ring or rings. Aryl groups include, for example, phenyl and naphthyl, including 1-naphthyl and 2-naphthyl.

"Heteroaryl" indicates a stable monodyl aromatic ring having the indicated number of ring atoms and containing from 1 to 3, or in some embodiments from 1 to 2, heteroatoms selected from N, O and S, the rest of the atoms being of the carbon ring, or a stable two-ring or tricyclic system containing at least one 5- to 7-membered aromatic ring containing from 1 to 3, or in some embodiments from 1 to 2, heteroatoms selected from N, O and S, the rest of the atoms of the carbon ring. Monodyl heteroaryl groups typically have 5 to 7 ring atoms. In some embodiments, the two-membered heteroaryl groups are 9 to 10 member heteroaryl groups. It is preferred that the total number of S and O atoms in the heteroaryl group is not greater than 2. Examples of heteroaryl groups include thienyl, pyridyl, pyrimidinyl and pyrrolyl.

"Heterocycloalkyl" is a saturated ring group, which has 1, 2, 3 or 4 heteroatoms independently selected from N, S and O, with the rest of the atoms in the carbon ring. Monodyl heterocycloalkyl groups typically have 4 to 6 ring atoms. Examples of heterocycloalkyl groups include morpholinyl, piperazinyl, piperidinyl and pyrrolinyl.

"Pharmaceutical compositions" are compositions that include at least one active ingredient, such as a compound or a salt of one of the active compounds disclosed herein, and at least one other substance, such as a support. The pharmaceutical compositions eventually contain one or more additional active ingredients. "Pharmaceutical combinations" or "combination therapy" refers to the administration of at least two active ingredients, which can be combined in a single dosage form or provided together in separate dosage forms, possibly with instructions that the active ingredients they must be used together to treat a disorder, such as hepatitis C, or a disorder associated with hepatitis C, or another viral infection, as described here.

"Pharmaceutically acceptable salts" include derivatives of the disclosed compounds in which the parent compound is modified by making non-toxic, inorganic and organic acid or base addition salts thereof. The salts of the present compounds can be synthesized from a parental compound containing a basic or acidic moiety by conventional chemical methods. In general, said salts can be prepared by reacting free acid forms of these compounds with a stoichiometric amount of the appropriate base (such as hydroxide, carbonate, bicarbonate or the like of Na, Ca, Mg or K), or by reacting Free base forms of these compounds with a stoichiometric amount of the appropriate acid. Such reactions are typically carried out in water or in an organic solvent, or in a mixture of both. The pharmaceutically acceptable salt may be in the form of a pure crystal, or in simple polymorphic form, or it may be used in a non-crystalline or amorphous, glassy or vftrea form, or a mixture thereof. In an alternative embodiment, the active ingredient can be provided in the form of solvate.

Examples of pharmaceutically acceptable salts include, but are not limited to, salts of mineral or organic acids from basic wastes, such as amines; alkaline or organic salts of acidic residues, such as carboxylic acids; and the like Pharmaceutically acceptable salts include conventional non-toxic salts and quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, as salts of conventional non-toxic acids, those derived from inorganic acids, such as hydrochloric, bromhydric, sulfuric, sulfamic, phosphoric, metric and the like are included; and salts prepared from organic acids, such as acetic, propionic, sucdnic, glycolic, stearic, lactic, malico, tartaric, dtrico, ascorbic, pamoic, maleic, hydroxyleleic, phenylactic, glutamic, benzoic, salidyl, mesophilic, spherical, Besophilic, sulfamyl, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isethionic, HOOC- (CH2) n-COOH, where n is 0-4, and the like. Lists of additional suitable salts, e.g., can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., P. 1418 (1985).

The term "support" applied to pharmaceutical compositions / combinations means a diluent, excipient or vehicle with which an active compound is provided.

A "pharmaceutically acceptable excipient" means an excipient that is useful in the preparation of a pharmaceutical composition / combination, which is generally safe, that is sufficiently non-toxic and that is not undesirable either biologically or in any other sense. A "pharmaceutically acceptable excipient", as used in the present application, includes both one and more than one of said excipients.

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A "therapeutically effective amount" of a pharmaceutical composition / combination of this invention means an effective amount, when administered to a patient, to provide a therapeutic benefit, such as an improvement of the symptoms, eg, an effective amount to reduce the Symptoms of a hepatitis C infection. For example, a patient infected with a hepatitis C virus may have elevated levels of certain liver enzymes, including AST and ALT. In certain embodiments, a therapeutically effective amount is, therefore, therefore, an amount sufficient to provide a significant reduction in the high levels of AST and ALT or an amount sufficient to provide a return of the levels of AST and ALT to the normal range.A therapeutically effective amount is also alternatively an amount sufficient to prevent a significant increase or significantly reduce the detectable level of viruses or antibodies for the virus in the patient's blood, serum or tissues. A method of determining the efficacy of treatment includes the measurement of HCV RNA levels by a conventional method for determining levels of vmco RNA, such as Roche's TaqMan assay. In certain preferred embodiments, the treatment reduces HCV RNA levels below the quantification Kmite (30 Ul / ml, as measured by Roche's TaqMan (R) test) or more preferably below the detection Kmite (10 Ul / ml, Roche's TaqMan).

A "patient" or "host" is a human or non-human animal that needs medical treatment. Medical treatment may include the treatment of an existing condition, such as a disease or disorder, prophylactic or preventive treatment or diagnostic treatment. Unless otherwise indicated, the patient or host is a human patient.

Highly active nucleoside derivatives

It has been surprisingly discovered that the uridine 2'-methyl-5'-deuterated phosphoramidate of Formula I, including Formula II, or Formula IIIA or IIIB, or a pharmaceutically acceptable salt thereof, where deuterium has an enrichment above of the protium of at least 90% (i.e. less than 10% of 1H hydrogen), and where R1 and R2 are independently deuterium or hydrogen, it is a superior NS5B inhibitor for the treatment of hepatitis C, or any other aqm disorder revealed. In one embodiment, R1 is deuterium and R2 is hydrogen.

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Formula II comprises mixtures of stereoisomers. For example, Formula II includes a 50/50 mixture of stereoisomers of Formula II, where the mixture comprises

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Therefore, in one embodiment, a method is provided for the treatment of a host infected with hepatitis C or a related disorder as described herein, which includes the administration of an effective amount of a compound of Formula I or II, or of a pharmaceutically acceptable salt thereof, optionally in a pharmaceutically acceptable carrier.

In an alternative embodiment, one or both of the 5'-deuterium (s) independently represent at least 50% enrichment. In another embodiment, the enrichment is independently at least 75% or 80%. In another embodiment, one or both of the 5'-deuterium (s) independently represent at least 90%, 95% or 98% enrichment.

In another embodiment, the nucleoside derivative of Formula I or II is administered as an R or S stereoisomer of phosphorus, which is at least in 90% pure form, and typically in 95, 98 or 99% pure form, or which is administered as a mixture of stereoisomers of chiral phosphorus centers, such as a 50/50 mixture of stereoisomers of chiral phosphorus centers, or a mixture in which the ratio of stereoisomers R to S in the chiral phosphorus center is 10:90 to 90:10 or from 30:70 to 70:30.

In another embodiment, an effective amount of a compound of Formula IIIA or IIIB or its pharmaceutically acceptable salt, optionally on a pharmaceutically acceptable carrier, is provided to a host in need of hepatitis C therapy, or other therapy as disclosed herein.

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OR

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Surprisingly, it has been discovered that deuterium at the 5 ’position of the nucleoside stabilizes the nucleoside derivative against dephosphorylation to the unwanted 5’-OH, 5’-deuterated nucleoside. This is surprising, since the atom / s of deuterium is not cleaved during dephosphorylation and does not bind to an atom that is cleaved during dephosphorylation. Therefore, 5’-deuterium can produce a significant effect on metabolism and efficacy through a remote and unexpectedly important effect of the secondary deuterium isotope. Such an important effect of the secondary deuterium isotope on the disease has not been previously reported in the literature.

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disassembly in position 5 '. By increasing the stability of the nucleoside 5'-monophosphate against dephosphorylation, an increase in the nucleoside active 5'-triphosphate pool is achieved, which may result in greater efficacy at a given oral dosage or equal efficiency using a lower dose of nucleoside. It can also have a significant effect on the half-life, and therefore on the pharmacokinetics, of the drug.

The present disclosure provides a significant improvement over the compounds disclosed in US Patent Application Publication US 2012/0071434; U.S.S.N. 13 / 36,435), published on March 22, 2012 and granted to Alios BioPharma, Inc., which describes phosphorothioamidate nucleosides for the treatment of vmcas diseases. It is known that the replacement of oxygen with sulfur in a nucleoside 5'-phosphate stabilizes the phosphate against nucleotidase or other hydrolyzing action, which forms the basis for the use of phosphorothioates and other thiophosphate derivatives in antisense and chemical chemistry. stabilization of aptamers, which was developed in the 80s. See, in general, among other references, Pearson, "Characterization of ectonucleotidases on vascular smooth-muscle cells", Biochem J. (1985), 230, 503-507. One of the compounds disclosed by Alios is:

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The monothiophosphate metabolite resulting from the cleavage of phosphorothioamidate 6037 in vivo is stabilized against further enzymatic destruction towards free 5'-hydroxynucleoside due to the presence of the non-natural stabilizing sulfur atom. Therefore, the stabilizing effect of the deuteration in the 5 'position is masked by sulfur. In addition, it is also known that nucleoside monothiophosphates are hydrolyzed to nucleoside monophosphates by the enzyme Hint1 (the enzyme that is also responsible for the production of Formula V monophosphate, as well as monothiophosphates, from their respective prodrugs), releasing H2S, which can cause physiological and pathogenic effects (see Ozga et al., J. Biol. Chem. 2010, 285, 40809).

A significant improvement provided by the present invention is the surprising discovery that 5'-deutation using a more natural phosphoramidate, that is, without sulfur, protects the monophosphate from further destruction of the free hydroxyl group in a way that minimizes toxicity and more closely mimics natural compounds. The fact that this is not an obvious invention is dramatically highlighted with a revision of the US Publication 2011/0251152 (U.S.S.N. 13 / 076.552), granted to Pharmasset, Inc., the company that Sovaldi developed. On page 16 of the publication, this nucleoside-experienced company described the use of deuteration in six different Sovaldi species, but I never consider putting deuterium at position 5 ', which is now determined to be the most important position.

Treatment methods

The disclosure provides a method for treating a host, typically a human, infected with hepatitis C, or another disorder described herein, with an effective amount of one of the highly active nucleospheric derivatives of Formula I, possibly as a pharmaceutically acceptable salt and eventually in a pharmaceutically acceptable carrier.

In another embodiment, an effective amount of one of the highly active nucleosphonic derivatives described herein may be used, possibly as a pharmaceutically acceptable salt and possibly in a pharmaceutically acceptable carrier to treat a host, typically a human, with a secondary condition associated with hepatitis C, including, but not limited to, the disorders described below in (i) to (viii).

This disclosure provides methods of treating a viral infection in a patient, including a hepatitis C infection, by providing an effective amount of a compound or a pharmaceutically acceptable salt of Formula I to the patient infected with a hepatitis C virus. a compound or a salt of Formula I as the only active ingredient, or it may be provided together with one or more additional active ingredients. In certain embodiments, the compound or salt of Formula I is administered together with an NS3 protease inhibitor, an NS5A inhibitor, an NS5B inhibitor or a combination of the foregoing.

An effective amount of a pharmaceutical composition / combination of the disclosure may be an amount sufficient to (a) inhibit the progression of hepatitis C; (b) cause a regression of hepatitis C infection; or (c) cause a cure for a hepatitis C infection, so that the HCV virus can no longer be detected or

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antibodies to HCV in the blood or plasma of a previously infected patient. An amount of an effective pharmaceutical composition / combination to inhibit progression or cause hepatitis C regression includes an amount effective to stop worsening hepatitis C symptoms or reduce symptoms experienced by a patient infected with the virus. Hepatitis C. Alternatively, a stop of the progression or regression of hepatitis C may be indicated by any of several markers for the disease. For example, a lack of increase or reduction in the viral load of hepatitis C or a lack of increase or reduction in the number of circulating antibodies to HCV in a patient's blood are markers of a stop in the progression or regression of Hepatitis C infection. Other markers for hepatitis C disease include aminotransferase levels, particularly the levels of liver enzymes AST and ALT. Normal levels of AST are 5 to 40 units per liter of serum (the liquid part of the blood) and normal levels of ALT are 7 to 56 units per liter of serum. These levels will typically be elevated in a patient infected with HCV. The regression of the disease is usually marked by the return of the levels of AST and ALT to the normal range.

In yet another embodiment, an effective amount of one of the highly active nucleosphonic derivatives described herein can be used, possibly as a pharmaceutically acceptable salt and possibly in a pharmaceutically acceptable carrier, as prophylaxis to protect or prevent a host, typically a human, which has a hepatitis C infection, so that it does not evolve to a secondary condition associated with hepatitis C. In an alternative embodiment, an effective amount of one of the highly active nucleosphonic derivatives described herein can be used, possibly as a pharmaceutically acceptable salt. , and possibly on a pharmaceutically acceptable support, to treat a secondary condition associated with hepatitis C, including, but not limited to, the disorders described below in (i) to (viii).

(i) Cryoglobulinemia, which is the production of abnormal antibodies (called cryoglobulins) due to stimulation of lymphocytes by the hepatitis C virus. These antibodies can be deposited in small blood vessels, thus causing inflammation of the vessels (vasculitis) in the tissues throughout the body, including the skin, joints and kidneys (glomerulonephritis).

(ii) Non-Hodgkin B-cell lymphoma associated with hepatitis C, which is considered to be caused by excessive stimulation of B lymphocytes by the hepatitis C virus, which results in abnormal lymphocyte reproduction.

(iii) Cutaneous conditions, such as lichen planus and porphyria cutanea tar, have been associated with hepatitis C infection.

(iv) Cirrhosis, which is a disease in which normal liver cells are replaced with scar or abnormal tissue. Hepatitis C is one of the most common causes of liver cirrhosis.

(v) Ascites, which is the accumulation of fluid in the abdominal cavity, commonly caused by liver cirrhosis, which may be caused by hepatitis C infection.

(vi) Hepatocellular carcinoma, 50% of whose cases in the US are currently caused by chronic hepatitis C infection.

(vii) Jaundice related to hepatitis C, which is a yellowish pigmentation caused by an increase in bilirubin.

(viii) Thrombocytopenia is often found in patients with hepatitis C and may be the result of an inhibition of bone marrow, a decrease in thrombopoietin production in the liver and / or an autoimmune mechanism. In many patients, as hepatitis C progresses, platelet count decreases and both bone marrow inhibition and anti-platelet antibodies increase. Other symptoms and disorders associated with hepatitis C that can be treated with an effective amount of a pharmaceutical composition / combination of the disclosure include decreased liver function, fatigue, flu-like symptoms: fever, chills, muscle aches, joint pain and aches headache, nausea, aversion to certain foods, unexplained weight loss, psychological disorders, including depression, and tenderness in the abdomen.

The active compounds presented here can also be used to increase the liver function generally associated with hepatitis C infection, for example, the function of synthesis, including protein synthesis, such as serum proteins (eg, albumine, coagulation, alkaline phosphatase, aminotransferases (eg, alanine transaminase, aspartate transaminase), 5'-nucleosidase, glutaminyl transpeptidase, etc.), bilirubin synthesis, cholesterol synthesis and bile acid synthesis; a liver metabolic function, including carbohydrate metabolism, amino acid and ammonia metabolism, hormone metabolism and lipid metabolism; the detoxification of exogenous drugs; and a hemodynamic function, including splanchnic and portal hemodynamics.

The pharmaceutical compositions / combinations disclosed herein are also useful for treating viral infections in patients other than a hepatitis C infection. In an alternative embodiment, the infection may be an RNA virus infection, such as a Togaviridae, Picornaviridae, Coronaviridae virus infection. or Flaviviridae. The disclosure includes a method of treating an infection with a Togaviridae, Picornaviridae virus,

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Coronaviridae or Flaviviridae by administering an effective amount of one of the active compounds disclosed herein to a subject infected with a togavirus, picornavirus, coronavirus or flavivirus. Flaviviridae virus infections include infections with the Flavivirus, Pestivirus and Hepacivirus viruses. Flavivirus infections include yellow fever, Dengue fever, West Nile virus, encephalitis, including St. Louis encephalitis, Japanese B encephalitis, California encephalitis, Central European encephalitis, spring encephalitis. Russian summer and Murray Valley encephalitis, Wesselsbron disease and Powassan disease. Pestivirus infections include diseases primarily of cattle, including swine fever in pigs, VDVB (bovine vmca diarrhea virus) in cattle and Border Disease virus infections. Hepacivirus infections include Hepatitis C and Canine Hepacivirus. Togavirus infections include Sindbis virus, eastern equine encephalitis virus, western equine encephalitis virus, Venezuelan equine encephalitis virus, Ross River virus, O'nyong'nyong virus, Chikungunya virus, Semliki Forest virus and Rubella virus. Picornavirus infections include infections with the Aphthovirus, Aquamavirus, Avihepatovirus, Cardiovirus, Cosavirus, Dicipivirus, Enterovirus, Erbovirus, Hepatovirus, Kobuvirus, Megrivirus, Parechovirus, Salivirus, Sapelovirus, Senecavirus, Teschovirus and Tremovirus. Coronavirus infections include infections with the genus Alphacoronavirus, Betacoronavirus (which includes severe acute respiratory coronavirus (SARS)), Gammacorouavirus and Deltacoronavirus. The disclosure includes in particular compositions containing a compound of the present disclosure useful for treating Dengue fever, West Nile fever, yellow fever or BVDV (bovine vmca diarrhea virus), and methods of treating these infections. by administering the compound to a patient infected with the virus.

The disclosure also includes the following treatment methods: (i) A method of treating a host affected by hepatitis C, consisting of administering an amount of effective treatment of a nucleoside or nucleotide that has deuterium with an enrichment of at least 50% in the 5 'position of the nucleoside or nucleotide. (ii) A treatment method as in method (i), where the nucleotide is not a thiophosphate or typhosphate prodrug. (iii) A method for the treatment of an affected host of a disorder that can be treated with an effective amount of a nucleoside or nucleotide, the improvement consisting in administering the nucleoside or nucleotide as a 5'-deuterated nucleoside or nucleotide. (iv) A method of treatment as in (i), where the nucleotide is not a thiophosphate or typhosphate prodrug. (v) A method of treatment as in (i), where the enrichment is at least 90%. (vi) A method of treatment as in (i), where there are two deuteria in the 5 'position. (vii) A method of treatment as in (iii), where the nucleotide is not a thiophosphate or typhosphate prodrug. (viii) A method of treatment as in (iii), where the enrichment is at least 90%. (viii) The method of claim 23, where there are two deuteria in position 5 '.

Combination therapy

The present disclosure also includes pharmaceutical compositions and combinations containing an active compound described herein and at least one additional active ingredient, as well as treatment methods consisting of administering said compositions to a patient infected with hepatitis C, or another disorder described herein. In certain embodiments, the additional active ingredient is an HCV NS3 protease inhibitor or an HCV NS5A inhibitor or other NS5B inhibitor.

In non-limiting embodiments, the active compound for HCV of the present disclosure may be administered in combination or alternating with one or more of the active compounds that are a caspase inhibitor, a cyclophilin inhibitor, a cytochrome P450 monooxygenase inhibitor, an entry inhibitor, a glucocorticoid, an HCV protease inhibitor, a hematopoietin, a homeopathic therapy, an immunomodulatory compound, an immunosuppressant, an interleukin, an interferon or an interferon enhancer, an IRES inhibitor, a monoclonal antibody or polyclonal, a nucleoside or nucleotide analog or prodrug, a non-nucleospheric inhibitor, an NS4B inhibitor, an NS5A inhibitor, an NS5B inhibitor, a P7 protein inhibitor, a polymerase inhibitor, an RNAi compound, a vaccine Therapeutics, a TNF agonist, a tubulin inhibitor, a sphingosine-1-phosphate receptor modulator or a TLR agonist.

Non-limiting examples of active ingredients in these categories are:

Caspase inhibitors: IDN-6556 (Idun Pharmaceuticals).

Cyclophilin inhibitors: for example, NIM811 (Novartis), SCY-635 (Scynexis) and DEBIO-025 (Debiopharm). Inhibitors of cytochrome P450 monooxygenase: ritonavir, ketoconazole, troleandomycin, 4-methylpyrazole, cyclosporine, clomethiazole, cimetidine, itraconazole, fluconazole, miconazole, fluvoxamine, fluoxetine, nefazodone, sertraline, indinavirin, savirinaprinavirin, navirinaprinavirine, navirine, navirine, navirine, navirine, navirine, navirine, navirine, navirine, navirine, navirine, navirine, navirine, navirine, navirine, navirine, navirine, navirine, navirine, navirine, navirine, navirine, navirine, navirine, navirine, navirine, navirine, pyrolvirine , erythromycin and VX-497 (Merimebodib). Preferred CYP inhibitors include ritonavir, ketoconazole, troleandomycin, 4-methylpyrazole, cyclosporine and clomethiazole.

Input inhibitors: ITX-5061 (iTherX).

Glucocorticoids: hydrocortisone, cortisone, prednisone, prednisolone, methylprednisolone, triamcinolone,

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parametasona, betamethasone and dexamethasone.

HCV protease inhibitors: for example, Sovaprevir and ACH-2684. ABT-450 (Abbott), ACL-181 and AVL-192 (Avila), BMS-032 (Bristol Myers Squibb), Boceprevir (Merck), danoprevir (Hoffman-La Roche and Genentech), GS-9256 (Gilead), GS -9451 (Gilead), Telaprevir (VX-950, Vertex), VX-985 (Vertex), Simeprevir (TMC435, Tibotec), Fosamprenavir (Amprenavir prodrug, Glaxo / Vertex), Indinavir (CRIXIVAN, Merck), TMC435350 (Tibote / Medivir), Faldaprevir (BI 201335. Boehringer Ingelheim), PhX-1766 (Phenomix), Vaniprevir (MK-7009, Merck), narlaprevir (SCH900518, Schering), MK-5172 (Merck).

Hematopoietin: hematopoietin-1 and hematopoietin-2. Other members of the hematopoietin superfamily, such as the various colony stimulating factors (e.g. G-CSF, GM-CSF, M-CSF), Epo and SCF (stem cell factor).

Homeopathic therapies: Milk thistle, silymarin, ginseng, glycyrrhizin, licorice root, schisandra, vitamin C, vitamin E, beta carotene and selenium.

Immunomodulatory compounds: thalidomide, IL-2, hematopoietins, IMPDH inhibitors, for example Merimepodib (Vertex Pharmaceuticals Inc.), interferon, including natural interferon (such as OMNIFErOn, Viragen and SUMIFERON, Sumitomo, a mixture of natural interferons), interferon natural alpha (ALFERON, Hemispherx Biopharma, Inc.), interferon alpha-nl lymphoblast cell (WELLFERON, Glaxo Wellcome), oral alpha interferon, Peg-interferon, Peg-interferon alpha 2a (PEGASYS, Roche), recombinant interferon alpha 2a ( ROFERON, Roche), inhaled interferon alfa 2b (AERX, Aradigm), Peg-interferon alfa 2b (ALBUFERON, Human Genome Sciences / Novartis, PEGINTRON, Schering), recombinant interferon alfa 2b (INTRON A, Schering), pegylated interferon alfa 2b ( PEGINTRON, Schering, VIRAFERONPEG, Schering), interferon beta-la (REBiF, Ares-Serono, Inc. and Pfizer), interferon alpha consensus (INFERGEN, Intermune), interferon gamma-1b (ACTIMMUNE, Intermune, Inc.), interferon alfa not pegylated, alpha inte rferon, and its analogs, and synthetic alpha thymosin 1 (ZADAXIN, SciClone Pharmaceuticals Inc.), and lamdba interferon (BMS).

Immunosuppressants: sirolimus (RAPAMUNE, Wyeth).

Interleukins: (IL-1, IL-3, IL-4, IL-5, IL-6, IL-10, IL-11, IL-12), LIF, TGF-beta, TNF-alpha) and other factors low molecular weight (eg, AcSDKP, pEEDCK, thymus hormones and mini-cytokines).

Interferon enhancers: EMZ702 (Transition Therapeutics).

IRES inhibitors: VGX-410C (VGX Pharma).

Monoclonal and polyclonal antibodies: XTL-6865 (HEPX-C, XTL), HuMax-HepC (Genmab), Immunoglobin for Hepatitis C (human) (CIVACIR, Nabi Biopharmceuticals), XTL-002 (XtL), Rituximab (RITUXAN, Gene / IDEC), GS-6624 (Gilead).

Nucleoside analogs: Sofosbuvir (PSI-7977, Pharmasset and Gilead), PSI-7851 (Pharmasset), PSI-7977 (Pharmasset), R7128 (mericitabine, Roche), R7348 (Roche), NM283 (valopicitabine, Idenix), GS- 6620 (Gilead), TMC- 649 (Tibotec), VX-135 (Vertex, Alios), ALS-2200 (Alios), IDX184 (Idenix), IDX21437 (Idenix), IDX21459 (Idenix), Lamivudine (EPIVIR, 3TC, GlaxoSmithKline ), MK-0608 (Merck), zalcitabine (HlVID, Roche US Pharmaceuticals), ribavirin (including COPEGUS (Roche), REBETOL (Schering), VILONA (ICN Pharmaceuticals) and VIRAZOLE (ICN Pharmaceuticals), isatoribine (Anadys Pharmaceuticals), ANA245 (Anadys Pharmaceuticals) and viramidine (ICN), a prodrug of ribavirin amidine, combinations of nucleoside analogs can also be used.

Non-nucleosphic inhibitors: PSI-6130 (Roche / Pharmasset), ABT-333 and ABT-072 (Abbott), delaviridine (RESCRIPTOR, Pfizer), PF-868554 (Pfizer), GSK-852 (GlaxoSmithKline), Setrobuvir (ANA-598 , Anadys), VX-222 (Vertex), BI-127 (Boehringer Ingelheim) and BMS-325 (Bristol Meyers).

NS4B inhibitors: clemizol (Eiger BioPharmaceuticals, Inc.).

NS5A inhibitors: Daclatasvir (BMS-790052, BMS), AZD-729 (Astra Zeneca); PPI-461 (Presidio), PPI-688 (Presidio), samatasvir (IDX719, Idenix), ledipasvir (GS-5885, Gilead), GS-5816 (Gilead), ombitasvir (ABT-267, AbbVie), GSK2336805 (GlaxoSmithKline) and elbasvir (MK-8742, Merck).

NS5B inhibitors: MBX-700 (Microbotix / Merck), RG-9190, VX-222 (Vertex) and BMS-791325 (Bristol Meyers Squibb).

Protein P7 inhibitor: amantadine (SYMMETREL, Endo Pharmaceuticals, Inc.).

Polymerase inhibitors: ANA598 (Anadys), Tegobuvir (GS 9190, Gilead).

RNA interference: SIRNA-034 RNAi (Sirna Therapeutics).

Therapeutic vaccines: IC41 (Intercell), Gl 5005 (Globeimmune), Chronvac-C (Tripep / lnovio).

TNF agonists: adalimumab (HuMIRA, Abbott), entanercept (ENBrEl, Amgen and Wyeth), infliximab (REMICADE, Centocor, Inc.).

Tubulin inhibitors: Colchicine.

Sphingosine-1-phosphate receptor modulators: FTY720 (Novartis).

TLR agonists: TLR7 agonist (Anadys Pharmaceuticals), CPG10101 (Coley) and TLR9 agonists, including CPG 7909 (Coley).

Vaccines: HCV / MF59 (Chiron), IC41 (Intercell).

For example, in some embodiments, the additional active ingredient is sovaprevir or ACH-2684 (HCV NS3 protease inhibitors) and / or an NS5a inhibitor.

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The disclosure includes compositions in which the additional active ingredient is

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NS3 protease inhibitors, useful in the pharmaceutical compositions and compositions described herein, have been previously disclosed, for example in US Patent No. 7,906,619, issued March 15,

2011, aqrn incorporated as a reference in its entirety for its teaching regarding 4-amino-4- oxobutane peptides. The '619 patent is particularly incorporated by reference in the Examples section starting at column 50 and extending to column 85, which discloses useful compounds in compositions / combinations with the compounds of Formula I described herein.

US Patent Application No. 2010-0216725, published on August 26, 2010, is hereby incorporated by reference in its entirety for its teaching as regards 4-amino-4-oxobutanophilic peptides. The '725 application is particularly incorporated as a reference in the Examples section beginning on page 22 and extending to page 100, which discloses useful compounds in compositions / combinations with the Formula I Compounds described herein.

Published US Patent Application No. 2010-0152103, published on June 17, 2010, is hereby incorporated by reference in its entirety as regards its analogical analogs of 4-amino-4-oxobutanophilic peptide analogs. The '103 application is particularly incorporated as a reference in the Examples section beginning on page 19 and extending to page 60, which discloses useful compounds in compositions / combinations with the Compounds of Formula I and II described herein. In particular, the compounds of Formula I and II disclosed herein may be used in combination with an NS3 protease inhibitor of the formulas shown below.

NS5A inhibitors, useful in the pharmaceutical compositions and compositions described herein have been previously disclosed. U.S. Patent Publication No. US-2012-0302528, published November 29,

2012, is hereby incorporated as a reference in its entirety for its teachings regarding NS5A inhibitors.

In certain embodiments, the deuterated nucleoside prodrug is

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The NS3 protease inhibitor is selected from

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15 Pharmaceutical compositions

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The disclosed compounds can be administered as the net chemical compound, but are preferably administered as a pharmaceutical composition. Accordingly, the disclosure provides pharmaceutical compositions containing a compound or a pharmaceutically acceptable salt of any of the active compounds described herein, together with at least one pharmaceutically acceptable carrier. The pharmaceutical composition / combination may contain a compound or a salt of any of the active compounds described herein as the only active ingredient, but, in another embodiment, it may also contain at least one

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additional active substance In certain embodiments, it is preferred that the additional active ingredient be an NS3 protease inhibitor or an NS5A or NS5B inhibitor. In certain embodiments, the pharmaceutical composition is in a dosage form containing from about 0.1 mg to about 2,000 mg, from about 10 mg to about 1,000 mg, from about 100 mg to about 800 mg, or from about 200 mg at about 600 mg of a compound of Formula I and possibly from about 0.1 mg to about 2,000 mg, from about 10 mg to about 1,000 mg, from about 100 mg to about 800 mg, or from about 200 mg to about 600 mg of an additional active ingredient in a unit dosage form. In certain embodiments, the active compound is administered in an oral dosage form, such as a pfldora, tablet or capsule, in an effective amount, which may, in some embodiments, be at least 10, 25, 50, 100, 150 , 200, 250, 300, 350 or 400 mg. The pharmaceutical composition may also include a molar ratio of a compound of Formula I and an additional active ingredient. For example, the pharmaceutical composition may contain a molar ratio of about 0.5: 1, about 1: 1, about 2: 1, about 3: 1 or about 1.5: 1 to about 4: 1, and the other active ingredient can be, for example, an NS3 protease inhibitor, an NS5A inhibitor or another NS5B inhibitor.

The compounds disclosed herein may be administered by any suitable means, including oral, topical, parenteral, inhalation or spray, sublingual, transdermal, buccal or transmucosal administration, rectally, as an ophthalmic solution, as an injection or by other means. , in dosage unit formulations containing conventional pharmaceutically acceptable carriers. The pharmaceutical composition can be formulated as any pharmaceutically useful form, e.g., as an aerosol, a cream, a gel, a pfldora, a capsule, a tablet, a syrup, a transdermal patch or an ophthalmic solution. Some dosage forms, such as tablets and capsules, are subdivided into unit doses of a suitable size containing appropriate amounts of the active components, e.g., an amount effective to achieve the desired objective.

The supports include excipients and diluents and must be of a sufficiently high purity and of a sufficiently low toxicity to be suitable for administration to the patient under treatment. The support can be inert or it can have pharmaceutical benefits by itself. The amount of support employed together with the compound is sufficient to provide a practical amount of material for administration per unit dose of the compound.

Support classes include, but are not limited to, binders, buffering agents, coloring agents, diluents, disintegrants, emulsifiers, flavorings, glidants, lubricants, preservatives, stabilizers, surfactants, tabletting agents and moisturizing agents. Some media can be included in more than one class; For example, vegetable oil can be used as a lubricant in some formulations and as a diluent in others. Examples of pharmaceutically acceptable carriers include sugars, starches, cellulose, tragacanth powder, malt, gelatin, talc and vegetable oils. Any active ingredients may be included in a pharmaceutical composition, which do not substantially interfere with the activity of the compound of the present disclosure.

Pharmaceutical compositions / combinations may be formulated for oral administration. These compositions contain between 0.1 and 99% by weight (% p) of a compound of Formula I and usually at least about 5% p of a compound of formula. Some embodiments contain from about 25% p to about 50% p, or from about 5% p to about 75% p of the compound of formula.

An effective amount of a pharmaceutical composition / combination of the disclosure may be a sufficient amount, for example, to (a) inhibit the progression of hepatitis C; (b) cause a regression of hepatitis C infection; (c) cause a cure for a hepatitis C infection, so that HCV virus or antibodies to HCV can no longer be detected in the blood or plasma of a previously infected patient; or (d) treat an HCV-associated disorder. An amount of an effective pharmaceutical composition / combination to inhibit progress or cause hepatitis C regression includes an amount effective to stop worsening hepatitis C symptoms or reduce symptoms experienced by a patient infected with the virus. Hepatitis C. Alternatively, a stop of the progression or regression of hepatitis C may be indicated by any of several markers for the disease. For example, a lack of increase or reduction in the viral load of hepatitis C or a lack of increase or reduction in the number of circulating antibodies to HCV in a patient's blood may be markers of a progression or regression arrest. of hepatitis C infection. Other markers for hepatitis C disease include levels of aminotransferases, particularly the levels of liver enzymes AST and ALT. These levels will typically be elevated in a patient infected with HCV. The regression of the disease is usually marked by the return of AST and ALT levels to the normal range.

The compound or the pharmaceutically acceptable salt of Formula I and at least one additional active ingredient may

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be: (1) co-formulated and administered or supplied simultaneously in a combined formulation; (2) administered alternating or in parallel as separate formulations; or (3) by any other combination therapy regimen known in the art. When administered in alternating therapy, the methods of the disclosure may include administration or supply of the compound or salt of any of the active compounds described herein and an additional active ingredient sequentially, eg, in solution, emulsion, suspension, tablets , separate pills or capsules, or by different injections in separate syringes. In general, during alternation therapy, an effective dosage of each active principle is administered sequentially, that is, in a serial manner, while, in simultaneous therapy, effective dosages of two or more active ingredients are administered together. Various intermittent combination therapy sequences can also be used.

The dosage frequency may also vary depending on the compound used and the particular disease treated. However, for the treatment of mayone of infectious disorders, a dosage regimen of 4 times a day or less is preferred, and a dosage regimen of 1 or 2 times a day is particularly preferred.

It will be understood, however, that the specific dose level for any particular patient will depend on a variety of factors, including the activity of the specific compound employed, age, body weight, general health, sex, diet, time. of administration, the route of administration and the rate of excretion, the combination of drugs and the severity of the particular disease in the patient undergoing therapy.

The pharmaceutical package may include an active compound or a salt as described herein in a container, together with instructions for using the compound to treat a patient suffering from hepatitis C infection.

Also included are packaged pharmaceutical compositions / combinations. Such packaged combinations include any of the active compounds described herein in a container together with instructions for using the combination for the treatment or prevention of a viral infection, such as a hepatitis C infection, in a patient.

The packaged pharmaceutical composition / combination may include one or more additional active ingredients. In certain embodiments, the additional active ingredient is an NS3 protease inhibitor, an NS5A inhibitor or another NS5B inhibitor.

The packaged pharmaceutical combination may include an active compound described herein or a salt.

pharmaceutically acceptable of an active compound described herein and the additional active ingredient provided simultaneously in a single dosage form, concomitantly in separate dosage forms or provided in separate dosage forms for separate administration for some amount of time that is within the time in the that both the active compound described here and the additional active ingredient are in the bloodstream of the patient.

The packaged pharmaceutical combination may include an active compound described herein or a salt.

pharmaceutically acceptable of an active compound described herein provided in a container with an additional active agent provided in the same container or in a separate one, with instructions for using the combination to treat an HCV infection in a patient.

Abbreviations

The following abbreviations are used in the chemical process and in the examples.

 Ac2O

 Acetic acid

 Acod

 Acetic acid, deuterated

 Ac.

 aqueous

 BuOH

 Butanol

 DCM

 Dichloromethane

 EtOAc

 Ethyl acetate

 IBX

 2-yodoxibenzoic acid

 MeOH

 Methanol

 MTBE

 Methyl tert-butyl ether

 PBS

 Phosphate buffered saline solution

 PDC

 Pyridinium dichromate

 TORCH

 Triethylamine

 THF

 Tetrahydrofuran

 ‘BuMgCl

 Tert-Butylmagnesium Chloride

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Preparation of highly active nucleoside derivatives

Procedures are provided for the preparation of the active compounds disclosed herein. As an example, the compound of Formula II can be prepared by the steps

image22

consisting of:

(i) the reaction of an amino ester (100), where the amino ester is isopropyl ester of L-alanine, with a dichlorophosphate (200), where the dichlorophosphate is phenoxy dichlorophosphate, to form a reaction mixture;

image23

(ii) the addition to the reaction mixture of (i) an arylhydroxyl or arylsulfhydryl, R-LH, where L is S or O, and R is an optionally substituted aryl, heteroaryl or heterocycloalkyl group, such as phenyl, pyrrole, pyridyl , pyridinyl or indole, or alternatively R-LH may be an N-hydroxyimide, such as N-hydroxysuccinimide or N-hydroxyphthalimide, and in certain embodiments R-LH is

image24

to form an intermediary (300)

image25

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Y

(iii) reaction of the intermediate (300) with a nucleoside (400)

image26

to form

image27

In certain embodiments, the intermediary (300) has the following stereochemistry

image28

and a Formula IIA product is produced.

image29

In certain embodiments, the amino ester (100) and dichlorophosphate (200) are combined at a temperature below -20 ° C, more preferably at a temperature of about -40 ° C to about -60 ° C.

In certain embodiments, triethylamine or other base is added to the mixture of the amino ester (100) and dichlorophosphate (200). In certain embodiments, the addition occurs in an organic solvent, such as dichloromethane, or other organic solvent, such as 2-methyltetrahydrofuran or tetrahydrofuran.

Arylhydroxyl or arylsulfhydryl is added to the reaction mixture formed by the combination of aminoester (100) and dichlorophosphate (200). In certain embodiments, the arylhydroxy or arylsulfhydryl is trichlorothiophenol, but it can also be substituted by other groups, such as nitrothiophenol, bromothiophenol, N-hydroxysuccinamide, N-hydroxyphthalimide and nitrohydroxypyridine. In certain embodiments, the arylhydroxyl or arylsulfhydryl is added as a solution in

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Four. Five

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dichloromethane or other organic solvent, such as 1-propanol, 2-methyltetrahydrofuran or tetrahydrofuran. In certain embodiments, the solution containing the arylhydroxyl or arylsulfhydryl also contains triethylamine or other base. After adding the arylhydroxyl or arylsulfhydryl to the reaction mixture formed by the combination of aminoester (100) and dichlorophosphate (200), the resulting solution can be heated to a temperature greater than 0 ° C, greater than 15 ° C and preferably up to from about 20 ° C to about 35 ° C, and it can be stirred at this temperature for a period of about 5 hours to about 30 hours and more preferably about 10 hours to about 20 hours or about 15 hours.

The reaction mixture formed by the addition of arylhydroxyl or arylsulfhydryl to aminoester (100) and dichlorophosphate (200) can be extracted with water, which is eventually saturated with salt, such as sodium bicarbonate or ammonium sulfate. The crude intermediate (300) obtained by drying the organic fraction can be purified by column chromatography, recrystallization or other suitable purification method. The desired isomer of the intermediate (300) can be obtained by dissolving the intermediate, preferably after purification, in ethyl acetate / heptane or another mixture of other polar non-polar / aprotic solvents, such as a mixture of heptane, cyclohexane, benzene (non-solvent polar) and THF, DMF or DCM (polar aprotic solvents), and seeding the solution with a small amount of the desired isomer of the intermediate (300). (This amount of seed of (300) may have been obtained by another method).

Nucleoside (400) can be suspended in a solvent, preferably a non-polar aprotic solvent, such as THF, DCM or DMF. The nucleoside suspension (400) in solvent may be cooled below 0 ° C, preferably below -10 ° C to about -40 ° C and preferably to about -20 ° C. Note that nucleoside 400 is labeled as Formula VI or as compound 9 somewhere else in the specification. For reasons of convenience, 100 to 400 are used in this discussion of the synthetic method.

The nucleoside suspension (400) in solvent can be added to a base, such as a Grignard reagent, for example tert-butyl MgCl, or other alkyl metal halide, at a temperature below 0 ° C, preferably below -10 ° C at about -40 ° C and preferably up to about -20 ° C. The nucleoside reaction mixture (400) in solvent and base is heated to above 0 ° C, and preferably up to about 20 ° C to about 30 ° C, and stirred for about 1 to about 5 hours, or for preferably about 2 to about 3 hours. The reaction mixture can then be cooled again to less than 0 ° C, preferably to less than -5 ° C to about -20 ° C and preferably to about -10 ° C. The intermediate (300), which may be optically pure, is added to the reaction mixture containing the nucleoside (400). The reaction mixture of intermediate (300) and nucleoside (400) is heated to more than 0 ° C, and preferably up to about 20 ° C to about 30 ° C, and stirred for at least 5 hours, preferably about 10 at about 20 hours, or preferably for about 15 hours. The reaction can be cooled to about 0 ° C and stopped with ammonium chloride or an acid, such as HCl or another acid capable of providing a pH of about 1 to 3 or preferably about 2. The resulting product can then be purified, a compound of Formula I, by extraction of the organic phase, column chromatography, HPLC, crystallization or any other suitable purification method.

In addition to a method of preparing a compound of the present disclosure, the disclosure also provides intermediates (300) useful for preparing a compound of Formula I:

image30

where -L-R has been previously defined. In certain embodiments, the intermediary is

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image31

In other embodiments, the intermediary is

image32

Examples

Example 1.2 - (((S) - (Perfluorophenoxy) (phenoxy) phosphoryl) amino) propanoate of (S) -isopropyl (Compound 1).

image33

The HCl salt of the isopropyl ester of L-alanine (160 g) is loaded into a 5-liter four-neck flask equipped with a mechanical stirrer, thermometer and addition funnel. Dichloromethane (1 L) is added to the flask and the suspension is cooled to -70 ° C, followed by the addition of triethylamine (200 g, 276 ml) over 45 min. A solution of phenyl dichlorophosphate (200 g) in dichloromethane (1 L) is added to the mixture over 2.5 h. The reaction mixture is stirred at this temperature for 90 min. more and then allowed to warm to 0 ° C over a period of 2 h and stir for 2 h at 0 ° C. A solution of 2,3,4,5,6-pentafluorophenol (174.4 g) in 400 ml of dichloromethane and a solution of triethylamine (105.4 g) in 200 ml of dichloromethane are added dropwise simultaneously. over a period of 1.2 h. The mixture is heated to room temperature and stirred overnight. The solid HCl salt of triethylamine is filtered and the cake is washed with dichloromethane (3x150 ml). The filtrate is concentrated under reduced pressure and the residue is triturated with MTBE (3.0 L). The white solid is removed by filtration. The cake is washed with MTBE (3x150 ml). The filtrate is concentrated and the resulting crude solid is triturated with 20% ethyl acetate in hexane (2.0 L). The solid is collected by filtration and washed with 10% NaHCO3 until the aqueous phase reaches a pH of 7, the solid is then washed with water and dried in a vacuum oven (55 ° C) for 28 h. The dry solid is mixed with 500 ml of heptane-EtOAc (5: 1) and stirred for 1 h. The solid is collected by filtration and washed with heptane-EtOAc (5: 1.2x80 ml), to give> 99% of a single isomer. The solid is dried to obtain compound 1.

In an alternative working procedure, the reaction mixture is filtered and the DCM layer is washed with an ac solution. 0.1 N NaOH, followed by water, is dried and evaporated to dryness. The residue is suspended in heptane / EtOAc (5: 1) and the solid is filtered. The solid is resuspended in heptane / toluene (85:15), to isolate the pure unique isomer.

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image34

MeO OMe

acetone, p-TSA

image35

PDC

f-BuOH

Ac20

ch2ci2

image36

image37

image38

TFA

H20

f-BuMgCI

THF

OR

image39

2,2-Dimethylpropane (140 ml) is added to 2'-C-methyluridine 2 (100 g) in acetone (700 ml). The resulting mixture is cooled in an ice bath for 30 min., P-toluenesulfonic acid (11 g) is then added and the reaction mixture is stirred at room temperature for 24 h. After completion of the reaction (monitored by HPLC), the reaction mixture is cooled in an ice bath for 30 min. and neutralized using fno potassium carbonate (12 g in 13 ml of water, pH 7-8). The solvent is removed under reduced pressure to dryness. THF (~ 500 ml) is added to the residue and solids are removed by filtration. The filtrate is coevaporated with silica gel and purified by chromatography on silica gel (5-15% MeOH in CHCF), to obtain compound 3. 1 H NMR (400 MHz, DMSO-d6, 300 K): 8 1 , 22 (s, 3H), 1.34 (s, 3H), 1.49 (s, 3H), 3.63 (dd, J = 12.0 Hz, 2.8 Hz, 1H), 3.69 (dd, J = 12.0 Hz, 3.1 Hz, 1H), 4.15 (m, 1H), 4.47 (d, J = 2.8 Hz, 1H), 5.25 (wide s, 1H), 5.63 (dd, J = 8.2 Hz, 2.3 Hz), 6.01 (s, 1H), 7.85 (d, J = 8.2 Hz, 1H), 11.37 (s, 1 H); LC-MS: 299 amu (M + 1).

Compound 4 is prepared following the procedure described by Corey et al. (J. Org. Chem. 1984, 49, 4735) with the modifications described below. 3 (50 g) in CH2Cl2 (1 L) PDC (126.1 g) is added to acetonide at room temperature, followed by Ac2O (171 g) and t-BuOH (248 g). The reaction temperature is maintained below 35 ° C during the reagents addition and then stirred at room temperature for 5 h. The reaction mixture was poured into K2CO3 aq. (250 g in 600 ml of H2O) and the organic layer is washed with CuSO4 (100 g in 1 l of H2O). Activated carbon (10 g) and silica gel (100 g) are added to the organic layer and stirred for 30 min. and it filters. The filtrate is evaporated and the residue is purified by silica gel chromatography (0-50% EtOAc in CHCF), to obtain 4. 1 H NMR (400 MHz, DMSO-de, 300 K): 8 1.25 (s , 3H), 1.41 (s, 3H), 1.46 (s, 9H), 1.48 (s, 3H), 3.31 (s, 1H), 4.61 (s, 1H), 4 , 79 (s, 1H), 5.70 (dd, J = 8.1 Hz, 2.0 Hz, 1H), 5.93 (wide s, 1H), 7.97 (d, J = 8.1 Hz, 1H), 11.41 (s, 1H); LC-MS: 369 amu (M + 1).

Lithium chloride (1.76 g) was stirred with NaBD4 (1.58 g) in EtOD for 1 h. Compound 4 (2.97 g) was added to this

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The solution was stirred at room temperature for 3 h and stopped with acetic acid-d, diluted with ethyl acetate, washed with saturated aqueous saline and evaporated to dryness. The residue was purified by silica gel chromatography to obtain the 5 5'-dihydrated compound.

Compound 5 (2.1 g) was treated with trifluoroacetic acid in the presence of water, to obtain nucleotide 6 5'-dihydrated. 1H NMR (400 MHz, CD3OD, 300 K): S 1.16 (s, 3H), 3.84 (d, J = 9.2 Hz, 1H), 3.91 (d, J = 9.2 Hz , 1H), 5.67 (d, J = 8.1 Hz, 1H), 5.96 (s, 1H), 8.14 (d, J = 8.1 Hz, 1H); 13C NMR (100 MHz, CD3OD, 300 K): S 20.2, 73.4,

80.0, 83.8, 93.2, 102.3, 142.5, 152.5, 166.0 (C-5 'of the ribose not observed).

Compound 6 (1.0 g) was converted into the phosphoramidate derivative 7 following the procedure described by Ross et al. (J. Org. Chem. 2011,76, 8311). 1H NMR (400 MHz, CD3OD, 300 K): S 1.15 (s, 3H), 1.21 (2 xd, J = 6.3 Hz, 6H), 1.35 (dd, J = 7.2 Hz, Jh p = 0.9 Hz, 3H), 3.79 (d, J = 9.2 Hz, 1H), 3.91 (dc, Jhp = 10.0 Hz, J = 7.2 Hz, 1H ), 4.08 (dd, J = 9.2 Hz, Jh, p = 2.2 Hz, 1H), 4.96 (septet, J = 6.3 Hz, 1H), 5.60 (d, J = 8.1 Hz, 1H), 5.96 (s, 1H), 7.20 (m, 1H), 7.26 (m, 2H), 7.37 (m, 2H), 7.67 (d , J = 8.1 Hz, 1H); 31P NMR (162 MHz, CD3OD, 300 K): S 3.8; LC-MS: 530 amu (M + 1).

Example 3. Preparation of 2 - (((SH ((2R, 3R, 4R, 5R) -5- (5-deutero-2,4-dioxo-3,4-dihydropmmidm-1 (2H) -N) -3 , (S) -isopropyl dihydroxy-4-methyltetrahydrofuran-2-yl) dideuteromethoxy) (phenoxy) phosphoryl) amino) propanoate

(Formula IIA, Compound 10).

image40

NaBD4 (7.96 g) is added portionwise to a cooled 70:30 v / v (5 ° C) mixture of EtOD / D2O (350 ml, 99% D) in a 1 L flask, followed by addition of ester of acetonide 4 (35 g) in portions (bubbles slowly). The resulting reaction mixture is stirred at room temperature for 3 h and then heated at 80 ° C for 1 d (1 H NMR spectroscopic analysis indicates> 85% deuterium incorporation at the 5-uracil position). The reaction mixture is filtered to remove solids and concentrated under reduced pressure to remove EtOD. Additional D2O is added and the resulting mixture is reheated to 95 ° C to increase the incorporation of deuterium at position 5 to> 98% (incorporation of D monitored by 1 H NMR spectroscopy). After completion of the reaction, half of the solvent is removed under reduced pressure, the mixture is cooled in an ice bath, AcOD (59 g) is added and the resulting mixture is stirred for 15-20 min. EtOAc (300 ml) and saturated aqueous saline solution (100 ml) are added, the organic layer is separated and the ac layer is reextracted. with EtOAc (150 ml), followed by THF (150 ml). The combined organic layers are concentrated, the resulting residue is dissolved in 10% MeOH and CHCl3 (300 ml), filtered, concentrated and purified by silica gel chromatography (ISCO, eluent DCM / MeOH), to obtain the acetonido 8 deuterado. 1 H NMR (400 MHz, DMSO-ds, 300 K): S 1.22 (s, 3H), 1.36 (s, 3H), 1.49 (s, 3H), 3.31 (s, 2H) , 4.14 (d, J = 2.8 Hz, 1H), 4.47 (d, J = 2.8 Hz, 1H), 5.21 (s, 1H), 6.01 (s, 1H) , 7.85 (s, 1 H), 11.36 (s, 1 H); LC-MS: 302 amu (M + 1).

Deuterated acetonide 8 (50 g) is added to a cooled solution (5 ° C) of 4 N HCl (250 ml) and stirred at room temperature for 3 h, during which time a white precipitate forms. The solvent is evaporated to dryness and water (100 ml) is added to the residue and stirred. The suspension is cooled to 5 ° C, stirred for 1 h and the white precipitate is collected by filtration. The solid is washed with fna water (75 ml) and dried, to obtain deuterated nucleoside 9. 1H NMR (400 MHz, CD3OD, 300 K): S 1.15 (s, 3H), 3.84 (d, J = 9.2 Hz, 1H), 3.91 (d, J = 9.2 Hz , 1H), 5.96

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(s, 1H), 8.14 (s, 1H); 13C NMR (100 MHz, CD3OD, 300 K): S 20.2, 73.4, 80.0, 83.8, 93.2, 142.4, 152.5, 166.0 (C-5 'of uracil ribose and C-5 not observed); LC-MS: 262 amu (M + 1).

Nucleoside 9 (37.3 g) in THF (750 ml) is cooled to -5 ° C. F-BuMgCl (1 M in THF, 430 ml) is added and the mixture is stirred for 30 min. at the same temperature The reaction mixture is stirred for another 30 minutes at room temperature, then cooled again to -5 ° C and a solution of 1 (129.5 g) in THF (650 ml) is added slowly. The reaction mixture is stirred at room temperature for 24 h, cooled to -5 ° C and cold 2N HCl (200 ml) is added, followed by stirring for 10 min. and the addition of an ac solution. saturated NaHCO3 (-250 ml, pH -8) and solid NaCl (50 g). The resulting mixture is stirred for 1 h and the organic layer is separated. The ac layer is removed. with THF (2 x 150 ml). All organic layers are combined and evaporated to dryness. The residue is partially purified on a short silica gel column (500 ml) (10-20% MeOH in CHCl3) and then further purified by silica gel chromatography (ISCO, 4 x 300 g cartridge, eluting with 0 -10% MeOH in CH2Ch), to obtain the title compound 10. 1H RmN (400 MHz, CD3OD, 300 K): S 1.15 (s, 3H), 1.21 (2 xd, J = 6, 3 Hz, 6H), 1.35 (dd, J = 7.2 Hz, Jh p = 0.9 Hz, 3H), 3.79 (d, J = 9.2 Hz, 1H), 3.91 ( dc, Jhp = 10.0 Hz, J = 7.2 Hz, 1H), 4.08 (dd, J = 9.2 Hz, Jh p = 2.3 Hz, 1H), 4.96 (septet, J = 6.3 Hz, 1H), 5.96 (s, 1H), 7.20 (m, 1H), 7.26 (m, 2H), 7.37 (m, 2H), 7.67 (s , 1 HOUR); 31P NMR (162 MHz, CD3OD, 300 K): S 3.8; LC-MS: 531 amu (M + 1).

Example 4. Alternative preparation of Formula IIA (Compound 10)

image41

Phenoxy dichlorophosphate (12.58 g) is added to a cold solution (-50 ° C) of isopropyl ester of L-alanine in CH2Cl2 (100 ml), followed by the addition of triethylamine (18.3 ml) in CH2Ch (36 ml) maintained at a temperature below -40 ° C. The reaction mixture is heated to room temperature slowly and stirred for 2 h and cooled again to -50 ° C. A solution of 2,4,5-trichlorothiophenol (12.74 g) in CH2Cl2 (20 ml) containing triethylamine (9.1 ml) is added. The reaction is heated to room temperature and stirred for 15 h. The reaction mixture is washed with water (-300 ml), followed by aq NaHCO3. saturated (-300 ml). The organic layer is separated, dried over Na2SO4 and evaporated to dryness under reduced pressure. The crude material is passed through a short silica column (CH2Ch / EtOAc from 0: 1 v / v to -1: 4 v / v) and the product is collected after evaporation of the solvent. The product is dissolved in 100 ml of 2.5% EtOAc in heptane and the solution is seeded with compound 11 (-10 mg) and stirred for 1 h at room temperature. The precipitate is collected by filtration, washed with a small amount. of the above solvent mixture of EtOAc / heptane and dried, to obtain 11 as a single isomer. 1 H NMR (400 MHz, CDCl 3, 300 K): S 1.24 (d, J = 6.3 Hz, 3 H), 1.26 (d, J = 6.3 Hz, 3 H), 1.41 (d , J = 7.0 Hz, 3H), 3.99-4.21 (m, 2H), 5.02 (septet, J = 6.3 Hz, 1H), 7.17-7.24 (m, 3H), 7.34 (m, 2H), 7.52 (s, 1H), 7.73 (d, Jh, p = 2.2 Hz, 1H); 31P NMR (162 MHz, CDCl3, 300 K): S 21.1.

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A suspension of 9 (1.0 g) in THF is cooled to -20 ° C and f-BuMgCl (11.6 ml, 1 M in THF) is added slowly, keeping the temperature of the mixture below -20 ° C. The reaction mixture is heated slowly to room temperature (~ 2 h), stirred for 2 h and then cooled to -10 ° C. Compound 11 (3.74 g) is added and the reaction mixture is heated to room temperature and stirred. After 15 h, the reaction mixture is cooled to 0 ° C, ac HCl is added. 2 N (up to pH ~ 2) and the solution is stirred for 30 min. at 0 ° C. Aqueous NaHCO3 (until pH ~ 8) is added, followed by NaCl (~ 3 g), and the mixture is stirred for 30 min. The organic layer is separated, dried and evaporated under reduced pressure. The crude material is purified by silica gel column chromatography (5% MeOH in CH2Ch), to obtain pure.

After isolating compound 11 by filtration, the filtrate, which is enriched in the other stereoisomer in the phosphorus, can be concentrated and purified by chromatographic techniques. This stereoisomer of 11 is treated with nucleoside 9 to obtain the compound 31 described in Example 12.

Example 5. Alternative preparation of Formula II (Compound 10)

image42

Compound 12 is prepared in an analogous manner to that described above in Example 4 for compound 11. Spectroscopic data for 12: 1 H NMR (400 MHz, CDCh, 300 K): S 1.19 (d, J = 6.3 Hz, 3H), 1.22 (d, J = 6.3 Hz, 3H), 1.39 (d, J = 6.7 Hz, 3H), 4.25-4.38 (m, 2H), 4.96 (septet, J = 6.3 Hz, 1H), 7.10 (d, J = 9.0 Hz, 1H), 7.19 (m, 1H), 7.25 (m, 2H), 7.34 (m, 2H), 8.50 (dd, J = 9.0 Hz, J = 2.9 Hz, 1H), 9.15 (d, J = 2.9 Hz, 1H); 31P NMR (162 MHz, CDCh, 300 K): S-3.4.

Nucleoside 9 is treated with compound 12 in an analogous manner to that described in Example 3, to obtain compound 10.

Compound 31 described in Example 12 can be prepared using the other stereoisomer of compound 12 in a manner analogous to that described in Example 4.

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1,2,3,5-tetra-0-benzoyl-2-C-methyl-pD-ribofuranose 13 (2.44 g) was treated with uracil-5-d1 14 (1.0 g), following the procedure described in Harry-O'kuru et al. (J. Org. Chem. 1997, 62, 1754) using non-deuterated uracil, to obtain protected nucleoside 15. Compound 15 was treated with NaOMe in MeOH, to obtain 2'-C-methyluridine-5-d1 (16) . 1H NMR (400 MHz, CD3OD, 300 K): S 1.16 (s, 3H), 3.78 (dd, J = 12.5 Hz, 2.6 Hz, 1H), 3.84 (d, J = 9.2 Hz, 1H), 3.92 (app d t, J = 9.2 Hz, 2.4 Hz, 1H), 3.98 (dd, J = 12.5 Hz, 2.2 Hz , 1H), 5.96 (s, 1H), 8.14 (s, 1H); 13C NMR (100 MHz, CD3OD, 300 K): S 20.2, 60.5, 73.4, 80.0, 83.9, 93.1, 142.4, 152.5, 166.0 (C -5 of the uracil not observed).

Compound 16 (0.7 g) was converted into the phosphoramidate derivative 17 in an analogous manner to that described in Example 3. 1 H NMR (400 MHz, CD 3 OD, 300 K): S 1.15 (s, 3 H), 1 , 21 (2 xd, J = 6.3 Hz, 6H), 1.35 (dd, J = 7.2 Hz, Jhp = 0.9 Hz, 3H), 3.79 (d, J = 9.2 Hz, 1H), 3.91 (dc, Jhp = 10.0 Hz, J = 7.2 Hz, 1H), 4.08 (m, 1H), 4.37 (ddd, J = 11.8 Hz, Jhp = 5.9 Hz, J = 3.7 Hz, 1H), 4.50 (ddd, J = 11.8 Hz, Jh p = 5.9 Hz, J = 2.0 Hz, 1H), 4, 96 (septet, J = 6.3 Hz, 1H), 5.96 (s, 1H), 7.20 (m, 1H), 7.26 (m, 2H), 7.37 (m, 2H), 7.67 (s, 1 H); 31P NMR (162 MHz, CD3OD, 300 K): S 3.8; LC-MS: 529 amu (M + 1).

Example 7. Preparation of 2 - (((S) - (((2R, 3R, 4R, 5R) -5- (6-deutero-2,4-dioxo-3,4-dihydropyrimidm-1 (2H) -N ) -3,4-dihydroxy-4-methyltetrahydrofuran-2-yl) methoxy) (phenoxy) phosphoryl) amino) propanoate of (S) -isopropyl (Compound 18).

image44

Compound 18 was prepared using uracil-6-d1 in a manner analogous to that described for compound 17 in Example 6. 1 H NMR (400 MHz, CD 3 OD, 300 K): S 1.15 (s, 3 H), 1, 21 (2 xd, J = 6.3 Hz, 6H), 1.35 (dd, J = 7.2 Hz, Jhp = 0.9 Hz, 3H), 3.79 (d, J = 9.2 Hz , 1H), 3.91 (dc, Jhp = 10.0 Hz, J = 7.1 Hz, 1H), 4.09 (m, 1H), 4.37 (ddd, J = 11.8 Hz, Jhp = 5.9 Hz, J = 3.7 Hz, 1H), 4.50 (ddd, J = 11.8 Hz, Jh p = 5.9 Hz, J = 2.0 Hz, 1H), 4.96 (septet, J = 6.3 Hz, 1H), 5.60 (s, 1H), 5.96 (s, 1H), 7.20 (m, 1H), 7.26 (m, 2H), 7 , 37 (m, 2H); 31P NMR (162 MHz, CD3OD, 300 K): S 3.8; LC-MS: 529 amu (M + 1).

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Compound 19 was prepared using uracil-5,6-d2 in a manner analogous to that described for compound 17 in Example 6. 1 H NMR (400 MHz, CD3OD, 300 K): S 1.15 (s, 3H), 1.21 (2 xd, J = 6.3 Hz, 6H), 1.35 (dd, J = 7.2 Hz, Jhp = 0.9 Hz, 3H), 3.79 (d, J = 9, 2 Hz, 1H), 3.91 (dc, Jhp = 10.0 Hz, J = 7.2 Hz, 1H), 4.09 (m, 1H), 4.37 (ddd, J = 11.8 Hz , Jh, p = 5.9 Hz, J = 3.7 Hz, 1H), 4.50 (ddd, J = 11.8 Hz, Jhp = 5.9 Hz, J = 2.0 Hz, 1H), 4.96 (septet, J = 6.3 Hz, 1H), 5.96 (s, 1H), 7.20 (m, 1H), 7.26 (m, 2H), 7.37 (m, 2H ); 31P NMR (162 MHz, CD3OD, 300 K): S 3.8; LC-MS: 530 amu (M + 1).

Example 9. Preparation of 2 - (((S) - (((2R, 3R, 4R, 5R) -5- (5,6-dideutero-2,4-dioxo-3,4-dihydropyrimidm-1 (2H) -N) -3,4- dihydroxy-4-methyltetrahydrofuran-2-yl) dideuteromethoxy) (phenoxy) phosphoryl) amino) propanoate of (S) -isopropyl

(Formula I, Compound 22).

image46

Nucleoside 20 was prepared by uracil-5,6-d2 in an analogous manner to that described for compound 16 in Example 6. Nucleoside 20 was converted to deuterated nucleoside 21 in an analogous manner to that described for compound 8 in Examples 2 and 3. Spectroscopic data for 21: 1H NMR (400 MHz, CD3OD, 300 K): S 1.15 (s, 3H), 3.84 (d, J = 9.2 Hz, 1H), 3, 91 (d, J = 9.2 Hz, 1H), 5.95 (s, 1H); 13C NMR (100 MHz, CD3OD, 300 K): S 20.2, 73.4,

80.0, 83.8, 93.1, 152.5, 166.0 (C-5 'of ribose, C-5 of uracil and C-6 of uracil were not observed). Nucleoside 21 was converted into the phosphoramidate derivative 22 in an analogous manner to that described in Example 3. Spectroscopic data for 22: 1 H NMR (400 MHz, CD 3 OD, 300 K): S 1.15 (s, 3 H), 1, 21 (2 xd, J = 6.3 Hz, 6H), 1.35 (dd, J = 7.2 Hz, Jhp = 0.9 Hz, 3H), 3.79 (d, J = 9.2 Hz , 1H), 3.91 (dc, Jhp = 10.0 Hz, J = 7.2 Hz, 1H), 4.08 (dd, J = 9.2 Hz, Jhp = 2.2 Hz, 1H), 4.96 (septet, J = 6.3 Hz, 1H), 5.95 (s, 1H), 7.20 (m, 1H), 7.26 (m, 2H), 7.37 (m, 2H ); 31P NMR (162 MHz, CD3OD, 300 K): S 3.8; LC-MS: 532 amu (M + 1).

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image47

Commercial 2-yodoxibenzoic acid (IBX, 3.54 g) is washed consecutively with acetonitrile (2 x 30 ml), acetone (2 x 20 ml) and Et2O (10 ml) and then thoroughly dried in vat before use . A mixture of 2 (0.894 g) and washed IBX (2.52 g) in anhydrous acetonitrile (90 ml) is refluxed for 2 h. The mixture is cooled and filtered to remove solids. The filtrate is concentrated and treated with CH2Cl2. The solids are removed again by filtration and the filtrate is concentrated under reduced pressure, to obtain 23 as a colorless foam.

Compound 23 (1.19 g) is dissolved in EtOD (15 ml) and NaBD4 (0.168 g) is added to this cloudy solution in portions at 0 ° C with stirring. The reaction mixture is stirred at room temperature for 2 h and then cooled to 0 ° C before adding an aqueous solution. saturated NH4Cl (1 ml) to stop the reaction. Saturated aqueous saline solution (30 ml) is added and the mixture is extracted with EtOAc (5 x 30 ml). The combined organic extracts are dried over anhydrous Na2SO4, filtered and evaporated to dryness under reduced pressure. The crude material is purified by silica gel column chromatography (10% MeOH in CH2Cl2 as eluent), to obtain 24.

Compound 25 is prepared in an analogous manner to that described for compound 8 in Examples 2 and 3. 1 H NMR (400 MHz, CD 3 OD, 300 K): 8 1.16 (s, 6 H), 3.76 (broad d , 2.6 Hz, 1H), 3.84 (d, J = 9.2 Hz, 2H), 3.92 (d of d, J = 9.2 Hz, 2.4 Hz, 2H), 3, 96 (wide d, J = 2.2 Hz, 1H), 5.67 (d, J = 8.1 Hz, 2H), 5.96 (s, 2H), 8.14 (2 xd, J = 8 , 1 Hz, 2H); 13C NMR (100 MHz, CD3OD, 300 K): 8 20.2, 60.2 (t, Jh, d = 21.3 Hz), 73.4, 80.0, 83.8, 93.1, 102 , 3, 142.5, 152.5,

166.0.

Phosphoramidate 26 is prepared analogously to that described for compound 10 in Example 3.

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30

image48

Compounds 27, 28 and 29 are prepared using methods analogous to those described in Example 3. Spectroscopic data for 29: 1 H NMR (400 MHz, CD 3 OD, 300 K): 8 1.16 (s, 6 H), 3.76 (wide d, 2.6 Hz, 1H), 3.84 (d, J = 9.2 Hz, 2H), 3.92 (d of d, J = 9.2 Hz, 2.4 Hz, 2H) , 3.96 (broad d, J = 2.2 Hz, 1H), 5.96 (s, 2H), 8.14 (2 xs, 2H); 13C NMR (100 MHz, CD3OD, 300 K): 8 20.2, 60.2 (t, Jh, d = 21.4 Hz), 73.4, 80.0, 83.8, 93.1, 142 , 4, 152.5, 166.0 (C-5 of the uracil not observed).

Example 12. Preparation of 2 - (((SH ((2R, 3R, 4R, 5R) -5- (5-deutero-2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl) -3 , (S) -isopropyl dihydroxy-4-methyltetrahydrofuran-2-yl) dideuteromethoxy) (phenoxy) phosphoryl) amino) propanoate

(Compound 31).

image49

The combined MTBE and EtOAc / hexanes washes of the synthesis of compound 1 in Example 1 are concentrated under reduced pressure, to obtain a mixture of 30 and 1. Nucleoside 9 is treated with this mixture in a manner analogous to that described in Example 3, to obtain phosphoramidate derivatives 31 and 10. Pure 31 is isolated by preparative HPLC. 1H NMR (400 MHz, CD3OD, 300 K): 8 1.15 (s, 3H), 1.24 (d, J = 6.3 Hz, 3H), 1.25 (d, J = 6.3 Hz , 3H), 1.34 (dd, J = 7.2 Hz, Jh, p = 1.0 Hz, 3H), 3.80 (d, J = 9.2 Hz, 1H), 3.92 (dc , Jh, p = 9.0 Hz, J = 7.2 Hz, 1H), 4.12 (dd, J = 9.2 Hz, Jh, p = 2.7 Hz, 1H), 5.00 (septet , J = 6.3 Hz, 1H), 5.99 (s, 1H), 7.22 (m, 1H), 7.26 (m, 2H), 7.39 (m, 2H), 7.72 (s, 1 H); 31P NMR (162 MHz, CD3OD, 300 K): 8 3.9; LC-MS: 531 amu (M + 1).

Example 13. Determination of nucleoside concentrations in human hepatocytes

For ease of reference, reference is made to the following Formulas in this example:

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image50

image51

Hepatocyte Preparation

Fresh hepatic hepatocytes plated in a 12-well or 6-well form were received (Life Technologies, Catalog # HMFN12 and # HMNF06). Upon receipt, the shipping medium was removed immediately and replaced with 1 ml or 2 ml of preheated culture medium (supplemented modified Chee medium; Xenotech LLC, Catalog # K2300) for the 12 and 6 well formats, respectively. The cells were acclimatized overnight at 37 ° C with a 5% CO2 atmosphere. The medium was aspirated from the 12 and 6 well plates and replaced with 1 ml or 2 ml, respectively, of fresh medium containing 20 | aM of Formula II, 20 | aM of Formula VII or solvent control (0.05 % of DMSO). Samples were incubated at 37 ° C, with a 5% CO2 atmosphere, in duplicate for Formula II and unique for Formula VII in each well format. The stability of the compounds in the absence of cells was also studied. At 24 hours, the medium was removed and frozen. The cells were washed twice with cold PBS. 70% cold methanol (0.75 ml or 1.5 ml, for 12 and 6 well formats, respectively) containing Formula X as internal standard to each well was added and the cells were gently removed from the plate by scraping . The recovered cells suspended in the methanol solution were aspirated into a vial and frozen at -80 ° C.

Extraction and analysis of LC-MS / MS of hepatocytes

The cell solutions were extracted overnight at -80 ° C in 70% methanol, removed from the freezer, thawed and vortexed. The tubes were centrifuged at 3,000 rpm for 15 minutes at 4 ° C. Supernatants were removed and analyzed by LC-MS / MS. Six concentrations of Formula II were prepared,

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Formula VII, Formula VI or Formula IX by serial dilution of reason 3 in DMSO. Kots of the compounds were added at the specified concentrations to 70% methanol containing internal standard. Two concentrations were also added to solutions of experiment cells incubated in the absence of compound. The samples were frozen at -80 ° C overnight and then thawed and vortexed. Samples were centrifuged at 3,000 rpm for 15 minutes. Supernatants were removed and analyzed by LC-MS / MS. The calibration concentrations were 5, 1.67, 0.556, 0.185, 0.0617 and 0.0206 | iM. Analytes were quantified using linear regression of standard calibration values with instrument response. The acceptance criteria used and the standard calibration concentrations were + 30% of the nominal concentration. Calibration standards that do not meet the criteria specified in the calibration curve were not used. Sample values were accepted when at least 66% of the standard concentrations during the operation were within 30% of the nominal. The "r" value required for the acceptance of the operation was> 0.98. Cell samples without an internal standard were analyzed due to only 81% extraction efficiency of the internal cell pattern while calibrating without cells; This gave a more precise determination of the concentrations.

Extraction and analysis of LC-MSlMS from hepatocyte media

The incubated hepatocyte media were removed from the freezer, thawed and vortexed. 2 parts of hepatocyte medium incubated with 1 part of internal standard containing acetonitrile were mixed and then centrifuged at 3,000 rpm for 15 minutes at 4 ° C. Supernatants were removed and analyzed by LC-MS / MS. The controls were six concentrations of Formula II, Formula VII, Formula VI or Formula IX prepared by serial dilution of ratio 3 in DMSO. Alfiquots of the compounds were added to fresh hepatocyte media, to obtain concentrations 5, 1.67, 0.556, 0.185, 0.0617 and 0.0206 µM of calibration medium. 2 parts of calibration medium were centrifuged mixed with 1 part of internal standard samples containing acetonitrile at 3,000 rpm for 15 minutes at 4 ° C. Supernatants were removed and analyzed by LC-MS / MS. Analyte concentrations in the samples were quantified using linear regression of standard calibration values with instrument response. The acceptance criteria used and the standard calibration concentrations were + 30% of the nominal concentration. Calibration standards that do not meet the criteria specified in the calibration curve were not used. Sample values were accepted when at least 66% of the standard concentrations during the operation were within 30% of the nominal. The "r" value required for the acceptance of the operation was> 0.98.

As the data in FIG. 1 and FIG. 2, there is more dephosphorylated nucleoside (i.e. unwanted 5'-OH nucleoside) in the samples incubated with the non-deuterated phosphoramidate than with the 5'-deuterated phosphoramidate. Specifically, using 20 | iM of Formula II or its non-deuterated Formula VII counterpart (12-well plate (1 ml) with hepatocytes seeded at a rate of 0.67 million cells per well for 24 hours) results in a concentration 1.9 times (medium, that is, extracellular concentration) and 2.9 times (cellular extract, that is, intracellular) greater than 2'-non-deuterated dephosphorylated methyluridine (Formula IX) compared to the resulting 5 'form -deuterated (Formula VI). The results of the incubation of 20 | iM of Formula II or its non-deuterated counterpart (6-well plate (2 ml) with hepatocytes seeded at the rate of 1.7 million cells per well for 24 hours) indicate a concentration 1.5 times (cellular extract, that is, intracellular) and 2.8 times (cellular extract, that is, intracellular) higher in a higher concentration of non-deuterated dephosphorylated 2'-methyluridine (Formula IV) compared to the resulting form 5 '-deuterated (Formula VI). Therefore, on average, the nucleotidase activity of hepatocytes results in approximately double the 5'-OH nucleoside produced when the 5 'position is not deuterated. This difference in the 5'-monophosphate pool available for triphosphate activation when the 5'-deuterated nucleoside derivative is used can have a significant effect on the efficacy, dosage, toxicity and / or pharmacokinetics of the drug.

Example 14. Levels of triphosphate (Formula IV) compared to the levels of triphosphate of VX-135-TP

In this Example, the results of three experiments comparing the levels of triphosphate of Formula IV with the level of triphosphate of VX-135 are described. Human hepatocytes were used according to the general methods described in Example 13. The concentrations of Formula IV produced in human hepatocytes (pmol of Formula IV / million cells) were determined at 2, 4, 8, 25 or 48 hours of incubation with 5 | iM of Formula II.

As compared to a candidate for a chemical test as will be described later in Example 14 and Figure 3, a poster presented by Alios (EASL 2013) indicates that the level of VX-135 triphosphate measured in human hepatocytes after 24 hours of incubation with 50 | iM of VX-135 it was 1,174 pmol / million cells. On the contrary, the level of Formula IV after 25 hours of incubation of human hepatocytes with 5 µM of Formula II, that is, a concentration ten times lower, is 486 pmol / million cells. Therefore, the amount of triphosphate produced by incubation of Formula II is 4 times higher (standardized dose) than the amount of triphosphate produced by VX-135. Although the precise structure of VX-135 is not currently known, it is about an urotine nucleotide analog prodrug of NS5B inhibitor.

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Example 15. Determination of the concentration of Formula IV from the dosage of Formula II

The ratio of the concentration of Formula IV (ng / ml) was determined as a result of the concentration of Formula II (| iM) in human hepatocytes. The general methods of Example 13 were used to determine the concentrations of compounds. Formula IV concentrations in human hepatocytes were determined after 24-hour incubations with 0.15, 0.45 and 1.35 µM of Formula II. The results were represented and linear regression was calculated using Microsoft Excel. As shown in Figure 4, there is a linear relationship to the concentrations studied for the dosage of Formula II and the resulting concentration of the active triphosphate compound (Formula IV).

Additionally, after incubation of Formula II at 50 nM in primary hepatocytes for 24 hours, the level of Formula IV varied from 9.2 to 16.2 pmol / million cells. These concentrations are 5 to 8 times higher than the concentrations obtained when Huh-luc / neo cells were incubated with 50 nM Formula II. Since Formula IV is the active species that inhibits the replication of HCV replicons in Huh-luc / neo cells, the predicted EC50 of Formula II in primary human hepatocytes signals 6.25-10 nM (versus HCV putative in primary hepatocytes) assuming that the linear relationship obtained in Figure 4 between Formula II and Formula IV continues at a lower concentration.

Example 16. Determination of the half-lives of Formula IV and GS-7977-TP

In this Example, the general methods of Example 13 were used to determine the half-lives of active triphosphate (Formula IV or GS-7977-TP) in human, dog, monkey and rat hepatocytes. To explain it briefly, Formula II or GS-7977 (Sovaldi) were added at selected concentrations to hepatocytes (human, dog, monkey and rat) and incubated at 37 ° C. Cell extracts of the supernatant of Formula IV or GS-7977-Tp (active triphosphate metabolites) were measured by high performance liquid chromatography with tandem mass spectrometric detection (LC-MS / MS). The human hepatocytes used for half-life determinations were 12-well cells of human hepatocytes and were seeded at a rate of 0.67 million cells per well. The canine hepatocytes used for the half-life determinations were 12-well cells of Beagle dog hepatocytes and were seeded at a rate of 0.67 million cells per well. The monkey hepatocytes used for half-life determinations were 12-well format cells of Cinomolgo monkey hepatocytes and were seeded at a rate of 0.9 million cells per well. The rat hepatocytes used for the half-life determinations were 12-well format cells of Sprague-Dawley rat hepatocytes (SD) and were seeded at a rate of 0.67 million cells per well. All cells were obtained from Life Technologies.

As shown in Figure 5, the half-life of Formula IV is greater than the half-life of Sovaldi triphosphate in hepatocytes of the four species. The greatest half-life was in human hepatocytes, followed by dog hepatocytes, then monkey and then rat. The half-lives range from 10 to 30 hours for Formula IV and from 8 to 23 hours for Sovaldi triphosphate.

Example 17. Levels of triphosphate (Formula IV and GS-7977-TP) in human hepatocytes

In this Example, the levels of triphosphate of Formula IV and GS-7977-TP were determined using the general methods described in Example 13. The results for the three experiments that determine the levels of triphosphate of Formula IV as described in the Table shown in Figure 3, are represented graphically and are shown here in Figure 6. The levels of the corresponding Sovaldi triphosphate (GS-79777) were also determined for comparison, and are shown in Figure 7. Summarizing, determined the concentrations of Formula IV or GS-7977-TP produced in human hepatocytes (pmol / million cells) at 2, 4, 8, 25 or 48 hours of incubation with 5 | iM of Formula II or GS7977 (Sovaldi), respectively.

In addition, as described in Example 17 and Figures 6 and 7, over a period of 48 hours, while the intracellular conversion to the corresponding Sovaldi triphosphate (GS-7977) measured in human hepatocytes is 2 times greater whereas that of the triphosphate derived from Formula II (that is, Formula IV), the concentration of Formula IV is still growing at 48 hours, while the concentration of Sovaldi metabolite triphosphate decreases from 24 to 48 hours. The increasing concentration of Formula IV, combined with its half-life of> 24 h, suggests the accumulation of the levels of Formula IV (the formula II triphosphate) in hepatocytes with repeated dosing. This trend, after an initial dosing ramp in vivo until acclimatization, can lead to a higher steady-state concentration of Formula IV in vivo for the triphosphate derived from Formula II than for the Sovaldi triphosphate. In addition, the intrinsic potency (the inhibitory effect on the RdRp activity of NS5B) of Formula IV (the formula II triphosphate) is 1.5 times better than the intrinsic potency of Solvadi triphosphate.

Claims (20)

5 10 fifteen 1. A compound of Formula I or a pharmaceutically acceptable salt thereof, where Formula I is image 1 where R1 and R2 are each hydrogen or D and each position represented as D has enrichment in deuterium of at least 50%. 2. A compound or salt of claim 1 of formula image2 3. A compound or salt of claim 2 of formula image3 4. A compound or salt of claim 2 of formula image4 5. A compound or salt of Claim 2, wherein each position represented as D has a deuterium enrichment of at least 90%. 5 10 fifteen twenty 25 30 35 40 6. A compound or salt of Claim 2, wherein each position represented as D has a deuterium enrichment of at least 95%. 7. A 50/50 mixture of stereoisomers of the compound of Claim 2, wherein the mixture consists of image5 8. A pharmaceutical composition that contains an active ingredient, wherein the active ingredient is a compound or salt of Claim 2, and also contains a pharmaceutically acceptable carrier. 9. The pharmaceutical composition of Claim 8, which contains one or more additional active ingredients. 10. The pharmaceutical composition of Claim 9, wherein the one or more additional active ingredients are an HCV NS3 protease inhibitor, an HCV NS5A inhibitor, an HCV NS5B inhibitor or a combination of the foregoing. 11. The pharmaceutical composition of Claim 9, wherein the one or more additional active ingredients are an inhibitor of NS5A and at least one of sovaprevir and ACH-2684. 12. A therapeutically effective amount of a compound or salt of Claim 2 for use in a method of treating an HCV infection in a patient. 13. A therapeutically effective amount of a pharmaceutical composition of Claim 8 for use in a method of treating an HCV infection in a patient. 14. A therapeutically effective amount of a first active ingredient and a therapeutically effective amount of one or more additional active ingredients for use in a method of treating an HCV infection in a patient, where the first active ingredient is a compound or salt of Claim 1 and the one or more additional active ingredients are selected from an NS3 inhibitor and an NS5A inhibitor. 15. A therapeutically effective amount of a compound or salt of Claim 2 for use in a method of treating a vmca infection by Flaviviridae in a patient, where the vmca infection by Flaviviridae is Dengue fever, the virus infection of the Western Nile, yellow fever or bovine vmca diarrhea virus infection. 16. A method for preparing a compound of formula image6 consisting of (i) the reaction of an amino ester (100), where the amino ester is isopropyl ester of L-alanine, with a dichlorophosphate (200), to form a reaction mixture; 5 10 fifteen twenty 25 image7 (ii) the addition to the reaction mixture of (i) of R-LH, where L is S or O and R is an aryl, heteroaryl or heterocycloalkyl group optionally substituted, or R-LH, where R-LH is an N -hydroxyimide, to form an intermediate (300) image8 Y (iii) the reaction of the intermediary (300) with a nucleoside (400) image9 to form image10 where each position D represented as D has a deuterium enrichment of at least 50%. 17. A method of Revindication 16 for preparing a compound of formula image11 where intermediary 300 has the structure image12 5 18. The method of Claim 16, wherein the amino ester (100) and dichlorophosphate (200) are combined at a temperature below -20 ° C and R-LH is selected from image13 The method of Claim 16, wherein the amino ester (100) and dichlorophosphate (200) are combined to a temperature from -40 ° C to about -60 ° C. 20. The method of Claim 18, wherein the mixture of aminoester (100) and dichlorophosphate (200) is added based. The method of Claim 20, wherein the base is triethylamine and the base addition to the mixture occurs in an organic solvent selected from dichloromethane, 2-methyltetrahydrofuran or tetrahydrofuran.

 Hepatocyte medium Hepatocyte cell extract adjusted to medium volume

 Parental compound

 % Parental left @ T24h Stability of the buffer Parental T24h 37 ° C Nucleoside (Formula IX or Formula VI) Parental conc. (H-M) Nucleoside (Formula IX or Formula VI)

 Conc. (| IM)

 % Dose 20 | iM Conc. (HM)% Dose 20 hM

 Formula VII

 0.82 97 8.4 42 <0.02 0.044 0.22

 Formula II

 0.41 97 4.4 22 <0.02 0.015 0.08

FIG. one 5

 Hepatocyte medium Hepatocyte cell extract adjusted to medium volume

 Parental compound

 % Parental left @ T24h Stability of the buffer Parental T24h 37 ° C Nucleoside (Formula IX or Formula VI) Parental Conc. (HM) Nucleoside (Formula IX or Formula VI)

 Conc. (HM)

 % Dose 20 hM Conc. (HM)% Dose 20 hM

 Formula VII

 4.9 97 5.2 26 <0.02 0.057 0.28

 Formula II

 3.9 97 3.4 17 <0.02 0.020 0.10

FIG. 2

 Time (h)

 Formula IV (pmol / million cells) VX-135-TP (pmol / million cells)

 repl rep2 rep3 Media + SD v

 2

 72.1 71.5 67.9 70.5 ± 2.25

 4

 130 129 129 129 ± 0.62

 8

 218 217 236 224 ± 10.6

 25

 506 465 488 486 ± 20.9 117.4 (Standard dose)

 48

 619 581 575 592 ± 24.3

FIG. 3 Conc. Formula IV (ng / ml) image14 FIG. 4

 Species

 Formula IV ti / 2 (h) GS-7977-TP ti / 2 (h)

 Human

 27.6 (26.0-29.4) to 22.6 (21.5-23.9)

 Dog

 29.6 (22.0-45.4) 17.7 (15.0-21.5)

 Monkey

 14.5 (11.8-18.9) 9.3 (7.9-11.4)

 Rat

 10.2 (8.6-12.5) 7.6 (6.4-9.3)

FIG. 5 image15 FIG. 6 </> <0 image16 FIG. 7